Difference between revisions of "Part:BBa K3114025"
 
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<partinfo>BBa_K3114025 short</partinfo>
 
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We were able to secrete 7-HCAR 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.]
 
We were able to secrete 7-HCAR 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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[[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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__NOTOC__
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<partinfo>BBa_K3114025 short</partinfo>
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===Usage and Biology===
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7-hydroxymethyl chlorophyll a reductase (7-HCAR) is an enzyme which catalyzes the reaction converting 7-hydroxymethyl chlorophyll a into chlorophyll a. (Meguro et al. 2011)
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BBa_K3114025 is a part consisting of multiple components of iGEM Calgary's 2019 Biobrick submission kit, as well as a few other well-characterized parts. The coding region contains the gene encoding for 7-HCAR[https://parts.igem.org/Part:BBa_K3114009(BBa_K3114009)].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)].
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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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===His Tag Purification===
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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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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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===Characterization===
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We were able to secrete 7-HCAR 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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<img src = "https://2019.igem.org/wiki/images/3/3c/T--Calgary--HCAR-PPH-SDS-PAGE-4-USE.png">
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<figcaption>10% SDS-PAGE HCAR and PPH Purification Confirmation. 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.</figcaption>
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===Sequences and Features===
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<partinfo>BBa_K3114025 SequenceAndFeatures</partinfo>
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===References===
 
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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.
 
  
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===Functional Parameters===
 
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<partinfo>BBa_K3114025 parameters</partinfo>
 
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===Sequences and Features===
 
===Sequences and Features===

Latest revision as of 10:47, 21 October 2019

7-Hydroxymethyl Chlorophyll A Reductase (7-HCAR) inducible circuit

Usage and Biology

7-hydroxymethyl chlorophyll a reductase (7-HCAR) is an enzyme which catalyzes the reaction converting 7-hydroxymethyl chlorophyll a into chlorophyll a. (Meguro et al. 2011)

BBa_K3114025 is a part consisting of multiple components of iGEM Calgary's 2019 Biobrick submission kit, as well as a few other well-characterized parts. The coding region contains the gene encoding for 7-HCAR[1].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.


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.

Characterization

We were able to secrete 7-HCAR 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.



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 1651
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 915
    Illegal AgeI site found at 339
    Illegal AgeI site found at 432
  • 1000
    COMPATIBLE WITH RFC[1000]


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.