Difference between revisions of "Part:BBa K3114027"
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===Usage and Biology===
 
===Usage and Biology===
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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).
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Pheophytinase (PPH) 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_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 PPH gene which encodes for pheopytinase[https://parts.igem.org/Part:BBa_K3114011 (BBa_K3114011)].A T7 promoter[https://parts.igem.org/Part:BBa_I719005 (BBa_I719005)]was used in conjunction with a strong ribosome binding site[https://parts.igem.org/Part:BBa_B0030 (BBa_B0030)].Following the coding region is iGEM Calgary's universal spacer sequence and 6XHis tag site[https://parts.igem.org/Part:BBa_K3114014 (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.
 
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 PPH gene which encodes for pheopytinase[https://parts.igem.org/Part:BBa_K3114011 (BBa_K3114011)].A T7 promoter[https://parts.igem.org/Part:BBa_I719005 (BBa_I719005)]was used in conjunction with a strong ribosome binding site[https://parts.igem.org/Part:BBa_B0030 (BBa_B0030)].Following the coding region is iGEM Calgary's universal spacer sequence and 6XHis tag site[https://parts.igem.org/Part:BBa_K3114014 (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.
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This part can be used for IPTG-inducible expression and secretion of the protein pheophytinase. 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).  
 
This part can be used for IPTG-inducible expression and secretion of the protein pheophytinase. 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).  
  
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===His Tag Purification===
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===Characterization===
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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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We were able to secrete PPH with the DsbA signal peptide using this genetic construct. The SDS-PAGE gel below shows the protein recovered from the periplasm for each of our genetic constructs as per our periplasmic protein isolation [https://2019.igem.org/Team:Calgary/Experiments protocol.]
  
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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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[[Image:Hcarpphgel.png|500px|thumb|center|Figure 1. 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), PPH - post loading fraction, PPH - elution fraction 1, PPH elution fraction 2, HCAR - post loading fraction, HCAR - elution fraction 1, HCAR elution fraction 2, pSB1A3 (plasmid control in BL21) - elution fraction 1, and pSB1A3 (plasmid control in BL21) - elution fraction 2. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa.]]
  
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We used thin-layer-chromatography to qualitatively assess the function of our PPH protein. We were able to achieve results in triplicate indicating that our protein is functional. More information on these results can be found [https://2019.igem.org/Team:Calgary/RepurposingChlorophyll#FinalResults (here)].
  
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[[Image:T-Calgary--PPHTLC.png|500px|thumb|center|Figure 1. Figure 8A and 8B: Thin Layer Chromatography Analysis of Pheophytinase Reactions. Samples were eluted using a methanol:hexane (70:30) solvent system on a silica plate. Lanes (read left to right) contain 24 hour reactions using Nickel-NTA purified PPH elution fraction 3, 2, 1, whole cell lysate (containing recombinant PPH) fraction 2, 1, pheophytin + reaction buffer solution, pheophytin + pheophorbide a + reaction buffer. Reaction buffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. This is one of three replicates.]]
  
 
===Sequences and Features===
 
===Sequences and Features===

Revision as of 11:01, 21 October 2019


Pheophytinase (PPH)

Usage and Biology

Pheophytinase (PPH) 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_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 PPH gene which encodes for pheopytinase(BBa_K3114011).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 pheophytinase. 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).

Characterization

We were able to secrete PPH with the DsbA signal peptide using this genetic construct. The SDS-PAGE gel below shows the protein recovered from the periplasm for each of our genetic constructs as per our periplasmic protein isolation protocol.

Figure 1. 10% SDS-PAGE was run at 100V for 15 minutes and then 180V for 35 minutes, then stained using Coomassie Blue. Lanes read left to right contain Color Prestained Protein Standard, Broad Range (11–245 kDa) as a ladder (NEB), PPH - post loading fraction, PPH - elution fraction 1, PPH elution fraction 2, HCAR - post loading fraction, HCAR - elution fraction 1, HCAR elution fraction 2, pSB1A3 (plasmid control in BL21) - elution fraction 1, and pSB1A3 (plasmid control in BL21) - elution fraction 2. NEB Ladder is shown on the left. HCAR is 58 kDa and PPH is 55.8kDa.

We used thin-layer-chromatography to qualitatively assess the function of our PPH protein. We were able to achieve results in triplicate indicating that our protein is functional. More information on these results can be found (here).

Figure 1. Figure 8A and 8B: Thin Layer Chromatography Analysis of Pheophytinase Reactions. Samples were eluted using a methanol:hexane (70:30) solvent system on a silica plate. Lanes (read left to right) contain 24 hour reactions using Nickel-NTA purified PPH elution fraction 3, 2, 1, whole cell lysate (containing recombinant PPH) fraction 2, 1, pheophytin + reaction buffer solution, pheophytin + pheophorbide a + reaction buffer. Reaction buffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. This is one of three replicates.

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 1579
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 267
    Illegal AgeI site found at 558
  • 1000
    COMPATIBLE WITH RFC[1000]

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.

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.

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.

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