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 our final culmination of our spectinomycin resistance gene pL1-aadA (BBa_K5078004), our pL1-NosZ-D denit 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 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 a restriction digestion 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 5337
Illegal PstI site found at 6823
Illegal PstI site found at 6916
Illegal PstI site found at 8496
Illegal PstI site found at 8983
Illegal PstI site found at 9192
Illegal PstI site found at 9362 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 6796
Illegal NheI site found at 10008
Illegal NheI site found at 10764
Illegal PstI site found at 5337
Illegal PstI site found at 6823
Illegal PstI site found at 6916
Illegal PstI site found at 8496
Illegal PstI site found at 8983
Illegal PstI site found at 9192
Illegal PstI site found at 9362
Illegal NotI site found at 11227
Illegal NotI site found at 11317 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 11638
Illegal BamHI site found at 12015
Illegal XhoI site found at 6643 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 5337
Illegal PstI site found at 6823
Illegal PstI site found at 6916
Illegal PstI site found at 8496
Illegal PstI site found at 8983
Illegal PstI site found at 9192
Illegal PstI site found at 9362 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 5337
Illegal PstI site found at 6823
Illegal PstI site found at 6916
Illegal PstI site found at 8496
Illegal PstI site found at 8983
Illegal PstI site found at 9192
Illegal PstI site found at 9362
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 9216
Illegal NgoMIV site found at 9584
Illegal NgoMIV site found at 9617
Illegal NgoMIV site found at 9686
Illegal AgeI site found at 8515
Illegal AgeI site found at 9602 - 1000COMPATIBLE WITH RFC[1000]
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