Difference between revisions of "Part:BBa K5078007"
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=pL1 PTC1 amiRNA=
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pL1-ptc-amiRNA (pL1-amiRNA) contains Btub promoter+5' UTR (BBa_K3002011), Ptc amiRNA which encodes for miRNA against Ptc mRNA (BBa_K5078002), and Psad Terminator (BBa_k3002002). pL1-amiRNA is part of the phosphorus side of our completed nutrient uptake plasmid (BBa_K5078009 and BBa_K5078010). pL1-amiRNA encodes for an artificial microRNA (amiRNA) that silences the expression of Phosphate Transport Channel 1 (PTC1) gene. pL1-amiRNA does this by causing a site specific cleavage of mRNA [1]. Specifically the mRNA responsible for PTC1 in Chlamydomonas reinhardtii. This prevent the formation of transport channels in the central vacuole. Causing phosphate (PO₄³⁻) to accumulate inside the cell and not leak back out into the environment.
  
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<img src="https://static.igem.wiki/teams/5078/psr1-amirna-chlamy.webp" width="400" height="auto"/><br>Figure 1. Ptc amiRNA in a level 1 plasmid for later golden gate assembly.
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<img src="https://static.igem.wiki/teams/5078/psr1-amirna-chlamy.webp" width="400" height="auto"/><br>Figure 1. Model of how amiRNA blocks PO₄³⁻ from leaving the C. reinhardtii. Along with modeling our Psr1 gene in C. reinhardtii (BBa_K5078003). Made with BioRender.
 
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<img src="https://static.igem.wiki/teams/5078/plasmid-pictures/ptc-amirna-l1-picture.webp" width="400" height="auto"/><br>Figure 1. Ptc amiRNA in a level 1 plasmid for later golden gate assembly.
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<img src="https://static.igem.wiki/teams/5078/plasmid-pictures/ptc-amirna-l1-picture.webp" width="400" height="auto"/><br>Figure 2. Plasmid diagram of pL1-ptc-amiRNA with Btub using benchling for modeling.
 
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===Plasmid Verification===
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<img src="https://static.igem.wiki/teams/5078/experiments/pl1-ptc-digest.webp" width="400" height="auto"/><br>Figure 1. Ptc amiRNA in a level 1 plasmid for later golden gate assembly.
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Successful transformation of pL1-P.stu into host bacterium can be determined by a restriction digestion with the restriction enzyme PaeI, with expected band lengths at 4295bp and 1288bp.
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<img src="https://static.igem.wiki/teams/5078/experiments/pl1-ptc-digest.webp" width="400" height="auto"/><br>Figure 3. A diagnostic digest of pL1-ptc-amiRNA with either a Psad terminator or a Btub terminator using PaeI on a 1% agarose gel. The restriction digest indicated that Btub colonies 4 was correctly transformed and was used for future experiments.  
 
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===Usage and Biology===
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This bar graph presents the results of a real-time quantitative PCR (qPCR) experiment measuring the expression levels of the PTC gene. The different bars represent the relative expression levels across various samples or conditions, with error bars indicating the variability within each group. This data provides insight into the differential expression of the PTC gene under the tested conditions and tells us in this experiment that Pstu.4 did the best.
  
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<img src="https://static.igem.wiki/teams/5078/results/ptc-rt-qpcr.webp" width="400" height="auto"/><br>Figure 4. Shows relative expression levels from a real-time qPCR experiment for PTC, with error bars indicating variability.
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<img src="https://static.igem.wiki/teams/5078/results/ptc-rt-qpcr.webp" width="400" height="auto"/><br>Figure 1. Ptc amiRNA in a level 1 plasmid for later golden gate assembly.
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====References====
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===Usage and Biology===
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[1] Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., Weigel, D. and Baulcombe, D. (2009), Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. The Plant Journal, 58: 165-174. https://doi.org/10.1111/j.1365-313X.2008.03767.x
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K5078000 parameters</partinfo>
 
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Revision as of 15:22, 29 September 2024

pL1 PTC1 amiRNA

pL1-ptc-amiRNA (pL1-amiRNA) contains Btub promoter+5' UTR (BBa_K3002011), Ptc amiRNA which encodes for miRNA against Ptc mRNA (BBa_K5078002), and Psad Terminator (BBa_k3002002). pL1-amiRNA is part of the phosphorus side of our completed nutrient uptake plasmid (BBa_K5078009 and BBa_K5078010). pL1-amiRNA encodes for an artificial microRNA (amiRNA) that silences the expression of Phosphate Transport Channel 1 (PTC1) gene. pL1-amiRNA does this by causing a site specific cleavage of mRNA [1]. Specifically the mRNA responsible for PTC1 in Chlamydomonas reinhardtii. This prevent the formation of transport channels in the central vacuole. Causing phosphate (PO₄³⁻) to accumulate inside the cell and not leak back out into the environment.


Figure 1. Model of how amiRNA blocks PO₄³⁻ from leaving the C. reinhardtii. Along with modeling our Psr1 gene in C. reinhardtii (BBa_K5078003). Made with BioRender.


Figure 2. Plasmid diagram of pL1-ptc-amiRNA with Btub using benchling for modeling.

Plasmid Verification

Successful transformation of pL1-P.stu into host bacterium can be determined by a restriction digestion with the restriction enzyme PaeI, with expected band lengths at 4295bp and 1288bp.


Figure 3. A diagnostic digest of pL1-ptc-amiRNA with either a Psad terminator or a Btub terminator using PaeI on a 1% agarose gel. The restriction digest indicated that Btub colonies 4 was correctly transformed and was used for future experiments.

Usage and Biology

This bar graph presents the results of a real-time quantitative PCR (qPCR) experiment measuring the expression levels of the PTC gene. The different bars represent the relative expression levels across various samples or conditions, with error bars indicating the variability within each group. This data provides insight into the differential expression of the PTC gene under the tested conditions and tells us in this experiment that Pstu.4 did the best.


Figure 4. Shows relative expression levels from a real-time qPCR experiment for PTC, with error bars indicating variability.

References

[1] Molnar, A., Bassett, A., Thuenemann, E., Schwach, F., Karkare, S., Ossowski, S., Weigel, D. and Baulcombe, D. (2009), Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. The Plant Journal, 58: 165-174. https://doi.org/10.1111/j.1365-313X.2008.03767.x

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 430
    Illegal PstI site found at 523
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 403
    Illegal PstI site found at 430
    Illegal PstI site found at 523
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 430
    Illegal PstI site found at 523
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 430
    Illegal PstI site found at 523
  • 1000
    COMPATIBLE WITH RFC[1000]