Part:BBa_K3114024

Chlorophyll B Reductase (CBR) inducible circuit

Usage and Biology

Chlorophyll B Reductase is a naturally occurring plant enzyme, encoded by the gene NYC1, which catalyzes the first step of chlorophyll b degradation (Horie et al. 2009). With the cofactor NADPH, chlorophyll b reductase converts chlorophyll b into 7-Hydroxymethyl Chlorophyll a.

BBa_K3114024 is a part consisting of multiple components of iGEM Calgary's 2019 Biobrick submission kit as well as other widely used Biobricks. The coding region consists of the gene for Chlorophyll b reductase(BBa_K3114008).A T7 promoter(BBa_I719005)was used in conjunction with a strong ribosome binding site(BBa_B0030).Following the coding region is iGEM Calgary's universal spacer sequence and 6XHis tag site(BBa_K3114014).The universal spacer was utilized in an attempt to mitigate any adverse effects that the his tag may have on the protein's function, as hypothesized in Meguro et al. 2011.

This part can be used for IPTG-inducible expression and secretion of the protein Chlorophyll B Reductase. This circuit contains the DsbA signal peptide. The DsbA signal peptide is a 19-amino acid sequence which targets fused proteins to the Sec secretion pathway in E. coli (Schierle et al., 2003). The fused protein is not folded until it is secreted to the periplasm. The signal peptide is cleaved after residue 19 by signal peptidase after secretion (recognition sequence: Ala-X-Ala).

Sequences and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1309
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Horie, Y., Ito, H., Kusaba, M., Tanaka, R., & Tanaka, A. (2009). Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. Journal of Biological Chemistry, 284(26), 17449-17456.

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.

Schierle, C. F., Berkmen, M., Huber, D., Kumamoto, C., Boyd, D., & Beckwith, J. (2003). The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), 5706–5713. https://doi.org/10.1128/JB.185.19.5706-5713.2003