Part:BBa_K5078009
pL2 -aadA-nosZ(P.stu)-Ptc amiRNA-Psr1
pL2-DeSlimer (Pstu)
pL2-aadA-nosZ(P. stutzeri)-Ptc amiRNA - Psr1 (pL2-DeSlimer(Pstu)) is a combination of four level 1 builds pL1-aadA (BBa_K5078004), pL1-nosZ(Pstu) (BBa_K5078005), pL1-Ptc amiRNA (BBa_K5078007)and pL1 Psr1 (BBa_K5078003). The goal of this build was to increase uptake of nitrate and phosphate in transformed C. reinhardtii. pL1-aadA was added in order to give C. reinhardtii spectinomycin resistance, which allowed for simple differentiation of transformed C. reinhardtii.
Figure 1. Plasmid diagram of pL2-DeSlimer(Pstu) 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 assembly of pL2-DeSlimer into host bacterium was determined by restriction digest with the restriction enzyme EcoRI, with 4 bands expected. Additionally, bacterial colonies should appear white in the present X-gal.
Figure 3. pL2-DeSlimer diagnostic digest using EcoRI on a 0.8% agarose gel. We selected colonies 6 and 9 for electroporation into chlamy.
Usage and Biology
Effectiveness of Ptc amiRNA
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 either DeSlimer (Ddenit or Pstu, as indicated). 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 effectiveness of the Ptc amiRNA in reducing expression of Ptc under the tested conditions and tells us in this experiment that Pstu.2 did the best.
Figure 4. Shows relative expression levels from a RT-qPCR experiment for Ptc, with error bars indicating variability.
Nutrient Uptake Experiments
To determine how well chlamy transformed with pL2-DeSlimer(Pstu) took up PO₄³⁻ and NO3 from its environment we performed nutrient uptake assays, along with the wild-type strains of C. reinhardtii 4039 and 1690, which were acquired from the Chlamy Collection (chlamycollection.org). These wild-type strains help to confirm how our plasmid was affecting nutrient uptake.
This plasmid was not used for our first nutrient uptake experiment. For our second experiment, we cultured C. reinhardtii in a mixed media of TAP and Allen media. Allen media has lower phosphate levels and uses nitrate for nitrogen content. The third experiment was with Allen media alone. For the second experiment, 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 for the third experiment, which prevented pellet formation and allowed for more light to reach the cells. In both of these experiments, we did not see a difference in nutrient uptake levels between our untransformed chlamy, and chlamy transformed with pL2-DeSlimer(Pstu).
Figure 4. These results show that our transformed chlamy does not take up more phosphate than the untransformed control in lower phosphate concentration media.
Figure 5. These results show that our transformed chlamy does not take up more nitrate than the untransformed control.
Figure 6. Graph of the maximum rates of phosphate uptake in all three of our phosphate experiments of C. reinhardtii transformed with pL2-Psr1, two untransformed wild-type strains, and C. reinhardtii transformed with our final plasmid (BBa_K5078009) in the different media types. 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. Unfortunately, chlamy transformed with pL2-DeSlimer did not have improved nutrient uptake. We speculate that this may be due to a slower rate of mitosis of the chlamy, possibly due to the insertion of such a large transgene construct.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3752
Illegal PstI site found at 5477
Illegal PstI site found at 6963
Illegal PstI site found at 7056
Illegal PstI site found at 8636
Illegal PstI site found at 9123
Illegal PstI site found at 9332 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 4514
Illegal NheI site found at 6936
Illegal NheI site found at 10148
Illegal NheI site found at 10904
Illegal PstI site found at 3752
Illegal PstI site found at 5477
Illegal PstI site found at 6963
Illegal PstI site found at 7056
Illegal PstI site found at 8636
Illegal PstI site found at 9123
Illegal PstI site found at 9332
Illegal NotI site found at 3356
Illegal NotI site found at 11367
Illegal NotI site found at 11457 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4731
Illegal BglII site found at 11778
Illegal BamHI site found at 3678
Illegal BamHI site found at 12155
Illegal XhoI site found at 3245
Illegal XhoI site found at 4799
Illegal XhoI site found at 6783 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3752
Illegal PstI site found at 5477
Illegal PstI site found at 6963
Illegal PstI site found at 7056
Illegal PstI site found at 8636
Illegal PstI site found at 9123
Illegal PstI site found at 9332 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3752
Illegal PstI site found at 5477
Illegal PstI site found at 6963
Illegal PstI site found at 7056
Illegal PstI site found at 8636
Illegal PstI site found at 9123
Illegal PstI site found at 9332
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 4286
Illegal NgoMIV site found at 9356
Illegal NgoMIV site found at 9724
Illegal NgoMIV site found at 9757
Illegal AgeI site found at 8655
Illegal AgeI site found at 9742 - 1000COMPATIBLE WITH RFC[1000]
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