Part:BBa_K5078003
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 with the constitutive Psad Promoter, in turn causing 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 should increase phosphate intake, thus decreasing the amount of excess nutrients from the environment.
Figure 1. Plasmid diagram of pL1-Psr using benchling for modeling.
Figure 2. Phosphate pathway. Psr1 is a transcription factor that works to activate 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.
Plasmid Verification
Successful transformation of pL1-Psr into host bacterium can be determined by a restriction digest 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; a luciferase reporter assay can also be performed to determine successful transformation.
Figure 3. pL1-Psr1 diagnostic digest using BsaI on an 8% agarose gel. The restriction digest indicated that colonies 4, 5, 7, and 8 showed the correct bands.
Usage and Biology
We determined that Psr1-nanoLuc was effectively integrated into the genome and expressed using a luciferase assay. Of the colonies tested, approximately 50% of them expressed the transgene.
Figure 4. Luciferase reporter assay for pL1-Psr in electroporated C. reinhardtii.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 895
Illegal PstI site found at 1382
Illegal PstI site found at 1591
Illegal PstI site found at 1761
Illegal PstI site found at 2238
Illegal PstI site found at 2673
Illegal PstI site found at 2841 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2407
Illegal NheI site found at 3163
Illegal PstI site found at 895
Illegal PstI site found at 1382
Illegal PstI site found at 1591
Illegal PstI site found at 1761
Illegal PstI site found at 2238
Illegal PstI site found at 2673
Illegal PstI site found at 2841
Illegal NotI site found at 3626
Illegal NotI site found at 3716 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4037
Illegal BamHI site found at 4414 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 895
Illegal PstI site found at 1382
Illegal PstI site found at 1591
Illegal PstI site found at 1761
Illegal PstI site found at 2238
Illegal PstI site found at 2673
Illegal PstI site found at 2841 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 895
Illegal PstI site found at 1382
Illegal PstI site found at 1591
Illegal PstI site found at 1761
Illegal PstI site found at 2238
Illegal PstI site found at 2673
Illegal PstI site found at 2841
Illegal NgoMIV site found at 1615
Illegal NgoMIV site found at 1983
Illegal NgoMIV site found at 2016
Illegal NgoMIV site found at 2085
Illegal NgoMIV site found at 2108
Illegal NgoMIV site found at 2127
Illegal NgoMIV site found at 2153
Illegal AgeI site found at 914
Illegal AgeI site found at 2001 - 1000COMPATIBLE WITH RFC[1000]
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