Part:BBa_K5078007
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 the 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 prevents 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 digest 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 colony 4 was correctly transformed and was used for future experiments.
Usage and Biology
This bar graph presents the results of a reverse-transcriptase quantitative PCR (RT-qPCR) experiment measuring the expression levels of the Ptc gene in two cultures of untransformed chlamy, and 3 strains of chlamy transformed with the Ptc amiRNA construct. The different bars represent the relative expression levels across various samples or conditions, with error bars indicating the variability within each group. The data suggests that for two of the transformed colonies, the amiRNA is effectively reducing levels of the Ptc mRNA as desired. This data provides insight into the differential expression of the PTC gene under the tested conditions and tells us in this experiment that Pstu.2 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 430
Illegal PstI site found at 523 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 403
Illegal PstI site found at 430
Illegal PstI site found at 523 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 250
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 430
Illegal PstI site found at 523 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 430
Illegal PstI site found at 523 - 1000COMPATIBLE WITH RFC[1000]
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