Difference between revisions of "Part:BBa K3114009"

Revision as of 23:51, 18 October 2019

7-Hydroxymethyl Chlorophyll A Reductase (7-HCAR)

Usage and Biology

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).

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.

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.

Sequences and Features

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.

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