Revision as of 22:31, 28 September 2024

pL1_Psr1

pL1_Psr1 consists of the of the Psad Promoter+5' UTR (BBa_K3002001), Psr1 CDS the phosphate starvation response 1 transcription factor (BBa_K5078008), nanoLuc a luciferase reporter gene (BBa_K2619004), and the Btub Terminator (BBa_K3002012). pL1-Psr is the phosphate half of our completed nutrient uptake plasmids (BBa_K5078009 and BBa_K5078010). pL1-Psr is responsible for inducing a phosphate (PO₄³⁻) starvation response in Chlamydomonas reinhardtii. Causing a luxury phosphate uptake from the environment. We do this by overexpressing Psr1 which causes C. reinhardtii to store PO₄³⁻ into phospholipids and nucleic acids, producing what is referred to as PolyP [1]. This PolyP then accumulates in acidocalcisomes [1]. By inducing a phosphate starvation response from C. reinhardtii we will cause the algae to take more phosphate from their environment, decreasing the amount of excess nutrients in their environment.

Figure 1. Plasmid diagram of pL1-Psr using benchling for modeling.

Plasmid Verification

Successful transformation of pL1-Psr into host bacterium can be determined by a restriction digestion with the restriction enzyme BsaI, with expected band lengths at 1438bp, 1918bp, and 5676bp. Additionally bacterial colonies should appear white in the present X-gal, and a luciferase reporter assay can be conducted as well to determine successful transformation.

Figure 2. pL1-Psri diagnostic digest using BsaI on a 8% agarose gel. The restriction digest indicated that colonies 4, 5, 7, and 8 showed the the correct bands.

Figure 3. Luciferase reporter assay for pL1-Psr in electroporated C. reinhardtii.

Figure 4. Phosphate pathway. 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 prevents the transporters from releasing phosphate. Psr1 is already found in chlamy.That each contain 6 copper atoms [1]. The dark blue is high confidence of the structure and red is low confidence.

Figure 5. Phosphate Assay Results Showing Phosphate Uptake in Experimental Condition 1

Structure simulation

For a structural simulation of Psr1 CDS see BBa_K5078008.

Figure 6. Structure prediction of nitrous oxide reductase by NosZ-P.stuzeri using AlphaFold. The structure has two identical 65.8kDa subunits that each contain 6 copper atoms [1]. The dark blue is high confidence of the structure and red is low confidence.

References

[1] Stephen P. Slocombe, Tatiana Zúñiga-Burgos, Lili Chu, Payam Mehrshahi, Matthew P. Davey, Alison G. Smith, Miller Alonso Camargo-Valero, & Alison Baker. (2023). Overexpression of PSR1 in Chlamydomonas reinhardtii induces luxury phosphorus uptake. Frontiers in Plant Science, 14. https://doi.org/10.3389/fpls.2023.1208168

Sequence and Features