Part:BBa_K5078011
pL2-Psr1
pL2-aadA-Psr1 (pL2-Psr1) is a combination of our two level 1 builds pL1-aadA (BBa_K5078004), and pL1 Psr1 (BBa_K5078003). The goal of this build was to see how well the Psr1 gene induced a phosphate (PO₄³⁻) starvation response in Chlamydomonas reinhardtii, before we insert it into our final nutrient uptake plasmids (BBa_K5078009 and BBa_K5078010). This way we can determine if our nitogen half of our final plasmids is affecting the PO₄³⁻ half. Additionally it helps us determine if our PTC amiRNA (BBa_K5078007) helps or hinders Psr1. pL1-aadA was added to pL1-Psr1 in order to give C. reinhardtii spectinomycin resistance. This way we can easily differentiate transformed C. reinhardtii.
Figure 1. Plasmid diagram of pL2-aadA-Psr1 using Benchling for modeling.
Plasmid Verification
Successful transformation of pL2-Psr1 into host bacterium was determined by a restriction digestion with the restriction enzyme BsaI, with expected band lengths at 7259bp and 4635bp. Additionally bacterial colonies should appear white in the presence of X-gal.
Figure 2. pL2-Psr1 diagnostic digest using BsaI on a 8% agarose gel. The restriction digest indicated that colonies 1, 2, 3, and 4 were successfully transformed.
Usage and Biology
Phosphate Uptake Experiments
To determine how well pL2-Psr1 took up PO₄³⁻ from its environment we performed several phosphate uptake assays with it, along with two wild type strains of C. reinhardtii 1690cc and 4039, which were acquired from the Chlamy Collection (chlamycollection.org). These wild type strains help to confirm that pL2-Psr1 was affecting how C. reinhardtii took up PO₄³⁻, and it wasn’t simply a part of the cell's natural abilities.
For our experiments we would culture C. reinhardtii in different media. The first experiment was with TAP media due to it naturally having a high amount of phosphate in it and uses ammonium for nitrogen content. The second experiment was with a mixed media of TAP and Allen media C. Allen media has lower phosphate levels and uses nitrate for nitrogen content. The third experiment was with Allan media. C. reinhardtii was cultured in 15 ml conical tubes, while this was a convenient way to house C. reinhardtii it also resulted in large pellets forming in the bottom of the tubes. To fix this problem C. reinhardtii was cultured in Erlenmeyer flasks, which prevented pellet formation and allowed for more light to reach the cells.
Figure 3. Results of our first experiment using TAP media compared to an untransformed C. reinhardtii control. The results indicate strongly that pL2-Psr1 causes an increased rate of phosphate uptake of C. reinhardtii.
Figure 4. Results of our second experiment using mixed TAP/Allen media compared to an untransformed C. reinhardtii control, and chlamy transformed with BBa_K5078009. In this experiment, there was no evidence that pL2-Psr1 causes an increased rate of phosphate uptake of C. reinhardtii.
Figure 5. Results of our second experiment using Allen media compared to an untransformed C. reinhardtii control, and chlamy transformed with BBa_K5078009. In this experiment, there was no evidence that pL2-Psr1 causes an increased rate of phosphate uptake of C. reinhardtii.
Figure 6. Graph of the results of all three of our phosphate experiments. Comparing how well pL2-Psr1, two untransformed wild type strains, and C. reinhardtii transformed with our final plasmid (BBa_K5078009). C. reinhardtii transformed with Psr1 only outperformed untransformed C. reinhardtii in TAP media, which had the highest phosphate concentrations of any of the media we used for the assays. We speculate that at lower levels of phosphate, untransformed C. reinhardtii may turn on expression of its own Psr1 gene, causing it to uptake as much phosphate as our transformed C. reinhardtii.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3329
Illegal PstI site found at 3816
Illegal PstI site found at 4025
Illegal PstI site found at 4195
Illegal PstI site found at 4672
Illegal PstI site found at 5107
Illegal PstI site found at 5275 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4841
Illegal NheI site found at 5597
Illegal PstI site found at 3329
Illegal PstI site found at 3816
Illegal PstI site found at 4025
Illegal PstI site found at 4195
Illegal PstI site found at 4672
Illegal PstI site found at 5107
Illegal PstI site found at 5275
Illegal NotI site found at 6060
Illegal NotI site found at 6150 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 6471
Illegal BamHI site found at 6848 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3329
Illegal PstI site found at 3816
Illegal PstI site found at 4025
Illegal PstI site found at 4195
Illegal PstI site found at 4672
Illegal PstI site found at 5107
Illegal PstI site found at 5275 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3329
Illegal PstI site found at 3816
Illegal PstI site found at 4025
Illegal PstI site found at 4195
Illegal PstI site found at 4672
Illegal PstI site found at 5107
Illegal PstI site found at 5275
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 4049
Illegal NgoMIV site found at 4417
Illegal NgoMIV site found at 4450
Illegal NgoMIV site found at 4519
Illegal AgeI site found at 3348
Illegal AgeI site found at 4435 - 1000COMPATIBLE WITH RFC[1000]
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