Part:BBa_K5078010
pL2 -aadA-nosZ(D.denit)-Ptc amiRNA-Psr
pL2-DeSlimer (Ddenit)
pL2-aadA-nosZ-D.denit-Ptc-amiRNA-Psr1 (De-Slimer D.den) is the final culmination of our spectinomycin resistance gene pL1-aadA (BBa_K5078004), our pL1-NosZ-D denit gene, a nitrous oxide reductase gene from Dechloromonas denitrificans codon optimized for expression in Chlamydomonas reinhardtii (BBa_K5078006), our pL1 ptc amiRNA, which silences the expression of phosphate transporters (PTC1) in C. reinhardtii (BBa_K5078007), and our pL1 Psr1, which induces a phosphate (PO₄³⁻) starvation response in C. reinhardtii (BBa_K5078003). De-Slimer D.den is a nutrient uptake plasmid.
Figure 1. Plasmid diagram of pL2-DeSlimer(Ddenit) using benchling for modeling.
Figure 2. This is a diagram of the Phosphate pathway and Nitrogen pathway in C. reinhardtii. Psr1 is a transcription factor that works in the storage of lipids involved with phosphate. This allows phosphate to accumulate within the cell. The amiRNA reduces the expression of transporters involved in releasing phosphate. Psr1 is already found in C. reinhardtii. C. reinhardtii will uptake nitrate from its environment as a nitrogen source and convert it into N₂O, but with our inserted NosZ gene it will convert the greenhouse gas N₂O into N₂. This diagram was made using BioRender.
Plasmid Verification
Successful transformation of De-Slimer D.den into host bacterium was determined by restriction digest with the restriction enzyme EcoRI-HF, with expected band lengths at 1231bp, 2416bp, 4538bp, and 9440bp Additionally, bacterial colonies should appear white in the present X-gal.
Figure 3| Diagnostic digest showing that colonies 16, 19, 21, and 29 had successfully assembled plasmids.
Usage and Biology
We performed a luciferase assay on chlamy colonies transformed with pL2-DeSlimer; of nearly 200 screened colonies, only several showed luciferase assay. This is likely due to the large size of the insert (12+kb).
Figure 4| Results of luciferase assay of 96 colonies tested.
We performed a series of PCR verification tests to verify that the entire insert had integrated into the chlamy genome. We found that colony 4 tested positive for each of the verification tests.
Figure 5| Results of first set of PCR verification tests.
Figure 6| Results of second set of PCR verification tests.
Unfortunately, so far we have been unable to detect the expression of nosZ-mCherry by immunofluorescence or Western blot. Experiments are continuing.
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 7. Shows relative expression levels from a real-time qPCR experiment for Ptc, with error bars indicating variability.
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