Difference between revisions of "Part:BBa K4452000"

 
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<partinfo>BBa_K4452000 short</partinfo>
 
<partinfo>BBa_K4452000 short</partinfo>
  
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PLT2 Promoter Arabidopsis
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<p>This promoter was identified for implementing root magnetotropism by overexpressing ferritin in statoliths of columella cells in Arabidopsis thaliana. </p>
  
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===Background===
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<p>To restore directional root growth in microgravity, Hopkins iGEM 2022 proposed that the existing gravitropic mechanisms can be engineered to respond to an artificial cue. We set out to engineer roots to grow in the direction of magnetic field gradients: magnetotropism.</p>
 +
<p>Plants sense gravity via statoliths—starch-laden organelles in root tip columella cells—which sediment due to their weight. Statolith sedimentation triggers changes in the efflux of auxin, a universal plant hormone that induces plant cell elongation. Polarized auxin accumulation along the upper and lower sides of roots causes differential elongation of cells, guiding root growth in the direction of gravity.</p>
 +
<p>We predicted that filling statoliths with iron-loading proteins, like ferritin, would allow the statoliths to move in response to a magnetic gradient. For our project we designed a genetic construct that allows for ferritin to be overexpressed in Arabidopsis and imported into statoliths.</p>
 +
 +
===Biology===
 +
 +
<p>We researched root-specific Arabidopsis promoters looking for ones that have specificity to columella cells of the root cap as those cells contain the statoliths that sense gravity and translate that signal into directional root growth [1].</p>
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 +
Candidates included:
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<ul>
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<li>
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PYK10-1457 promoter (Part:BBa_K2286005), a seedling and root specific promoter which was engineered from Pyk10 to increase strength by several times
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</li>
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<li>
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RCH1 Promoter, ROOT CLAVATA HOMOLOG1 (RCH1) promoter is generally highly active in the root tip, but not specific to the columella cells [2]
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</li>
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<li>
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PIN7 Promoter, active in the columella with off-target activity in stele, but has less activity in stele when root is differentiated [3]
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</li>
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<li>
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FEZ Promoter,  preferentially expressed in columella cells of root, but sometimes not active in the columella cells but instead active in the lateral root cap [3]
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</li>
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<li>
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PLT2 Promoter, expresses the PLT2 gene of the PLETHORA (PLT) protein family in the columella and meristem sections of roots [4, 5, 6]
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</li>
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</ul>
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 +
<p>We did not pursue PYK10-1457 promoter and RCH1 Promoter because they were not specific enough to columella cells.  We noted that PIN genes are responsive to changes in the auxin maximum. Since statolith sedimentation is supposed to impact the auxin maximum, using PIN7 promoter may inadvertently make a positive feedback loop between statolith sedimentation and ferritin expression, and we are unsure how this would affect magnetotropism. Therefore we eliminated PIN7 as a promoter candidate.</p>
 +
 +
<p>We decided to use the PLT-2 promoter, specifically the shorter 1.3 kb version (instead of the longer 5.8kb version) because the sequence’s shorter length makes it easier to assemble.
 +
</p>
  
  
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<partinfo>BBa_K4452000 parameters</partinfo>
 
<partinfo>BBa_K4452000 parameters</partinfo>
 
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===References===
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[1] Singh, Manjul et al. "Striking the right chord: signaling enigma during root gravitropism." Frontiers in Plant Science 8 (2017): 1304. https://doi.org/10.3389/fpls.2017.01304
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 +
[2] Li, Huchen et al. “Plant-Specific Histone Deacetylases HDT1/2 Regulate GIBBERELLIN 2-OXIDASE2 Expression to Control Arabidopsis Root Meristem Cell Number.” The Plant cell vol. 29,9 (2017): 2183-2196. https://doi.org/10.1105/tpc.17.00366
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[3] Marquès-Bueno, Maria Del Mar et al. “A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.” The Plant journal : for cell and molecular biology vol. 85,2 (2016): 320-333. https://doi.org/10.1111/tpj.13099
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[4] Santuari, Luca et al. “The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.” The Plant cell vol. 28,12 (2016): 2937-2951. https://doi.org/10.1105/tpc.16.00656
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[5] Galinha, C., Hofhuis, H., Luijten, M. et al. “PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development”. Nature 449, 1053–1057 (2007). https://doi.org/10.1038/nature06206
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[6] Aida, Mitsuhiro et al. “The PLETHORA Genes Mediate Patterning of the Arabidopsis Root Stem Cell Niche.” Cell vol. 119, 1 (2004): 109-120. https://doi.org/10.1016/j.cell.2004.09.018

Latest revision as of 06:11, 10 October 2022


PLT2 promoter

This promoter was identified for implementing root magnetotropism by overexpressing ferritin in statoliths of columella cells in Arabidopsis thaliana.

Background

To restore directional root growth in microgravity, Hopkins iGEM 2022 proposed that the existing gravitropic mechanisms can be engineered to respond to an artificial cue. We set out to engineer roots to grow in the direction of magnetic field gradients: magnetotropism.

Plants sense gravity via statoliths—starch-laden organelles in root tip columella cells—which sediment due to their weight. Statolith sedimentation triggers changes in the efflux of auxin, a universal plant hormone that induces plant cell elongation. Polarized auxin accumulation along the upper and lower sides of roots causes differential elongation of cells, guiding root growth in the direction of gravity.

We predicted that filling statoliths with iron-loading proteins, like ferritin, would allow the statoliths to move in response to a magnetic gradient. For our project we designed a genetic construct that allows for ferritin to be overexpressed in Arabidopsis and imported into statoliths.

Biology

We researched root-specific Arabidopsis promoters looking for ones that have specificity to columella cells of the root cap as those cells contain the statoliths that sense gravity and translate that signal into directional root growth [1].

Candidates included:

  • PYK10-1457 promoter (Part:BBa_K2286005), a seedling and root specific promoter which was engineered from Pyk10 to increase strength by several times
  • RCH1 Promoter, ROOT CLAVATA HOMOLOG1 (RCH1) promoter is generally highly active in the root tip, but not specific to the columella cells [2]
  • PIN7 Promoter, active in the columella with off-target activity in stele, but has less activity in stele when root is differentiated [3]
  • FEZ Promoter, preferentially expressed in columella cells of root, but sometimes not active in the columella cells but instead active in the lateral root cap [3]
  • PLT2 Promoter, expresses the PLT2 gene of the PLETHORA (PLT) protein family in the columella and meristem sections of roots [4, 5, 6]

We did not pursue PYK10-1457 promoter and RCH1 Promoter because they were not specific enough to columella cells. We noted that PIN genes are responsive to changes in the auxin maximum. Since statolith sedimentation is supposed to impact the auxin maximum, using PIN7 promoter may inadvertently make a positive feedback loop between statolith sedimentation and ferritin expression, and we are unsure how this would affect magnetotropism. Therefore we eliminated PIN7 as a promoter candidate.

We decided to use the PLT-2 promoter, specifically the shorter 1.3 kb version (instead of the longer 5.8kb version) because the sequence’s shorter length makes it easier to assemble.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 95
    Illegal BglII site found at 527
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Singh, Manjul et al. "Striking the right chord: signaling enigma during root gravitropism." Frontiers in Plant Science 8 (2017): 1304. https://doi.org/10.3389/fpls.2017.01304

[2] Li, Huchen et al. “Plant-Specific Histone Deacetylases HDT1/2 Regulate GIBBERELLIN 2-OXIDASE2 Expression to Control Arabidopsis Root Meristem Cell Number.” The Plant cell vol. 29,9 (2017): 2183-2196. https://doi.org/10.1105/tpc.17.00366

[3] Marquès-Bueno, Maria Del Mar et al. “A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.” The Plant journal : for cell and molecular biology vol. 85,2 (2016): 320-333. https://doi.org/10.1111/tpj.13099

[4] Santuari, Luca et al. “The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots.” The Plant cell vol. 28,12 (2016): 2937-2951. https://doi.org/10.1105/tpc.16.00656

[5] Galinha, C., Hofhuis, H., Luijten, M. et al. “PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development”. Nature 449, 1053–1057 (2007). https://doi.org/10.1038/nature06206

[6] Aida, Mitsuhiro et al. “The PLETHORA Genes Mediate Patterning of the Arabidopsis Root Stem Cell Niche.” Cell vol. 119, 1 (2004): 109-120. https://doi.org/10.1016/j.cell.2004.09.018