Revision as of 16:04, 1 October 2024

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 see how well chlamy transformed with this plasmid increased their uptake of nitrate and phosphate from the media. pL1-aadA was added in order to give C. reinhardtii spectinomycin resistance. This way we can easily differentiate 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 a restriction digestion 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 electropoation into chlamy.

Usage and Biology

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,long with the wild type strains of C. reinhardtii 4039, which were acquired from the Chlamy Collection (chlamycollection.org). These wild type strains help to confirm that our inserted plasmidwas affecting how C. reinhardtii took up the nutrients, and it wasn’t simply apart of the cell natural abilities.

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 untransformed chlamy does not take up more phosphate than untransformed control.

Figure 5. These results show that our untransformed chlamy does not take up more nitrate than untransformed control.

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. 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 insertion of such a large transgene construct.

Sequence and Features