Difference between revisions of "Part:BBa K5078009"

 
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<partinfo>BBa_K5078009 short</partinfo>
 
<partinfo>BBa_K5078009 short</partinfo>
  
hwoejls dfshgoerhtoelrlv
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=pL2-DeSlimer (Pstu)=
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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.
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<img src="https://static.igem.wiki/teams/5078/plasmid-pictures/deslimer-pstu-picture.webp" width="400" height="auto"/><br>Figure 1. Plasmid diagram of pL2-DeSlimer(Pstu) using benchling for modeling.
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===Plasmid Verification===
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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.
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<img src="https://static.igem.wiki/teams/5078/experiments/digest-of-deslimer-pstu.webp" width="400" height="auto"/><br>Figure 2. pL2-DeSlimer diagnostic digest using EcoRI on a 0.8% agarose gel. We selected colonies 6 and 9 for electropoation into chlamy.
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===Nutrient Uptake Experiments===
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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.
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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).
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<img src="https://static.igem.wiki/teams/5078/results/exp-2-phosphate-assay.webp" width="600" height="auto"/><br>Figure 3. These results show that our untransformed chlamy does not take up more phosphate than untransformed control.
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<img src="https://static.igem.wiki/teams/5078/results/exp-2-nitrate-assay.webp" width="600" height="auto"/><br>Figure 4. These results show that our untransformed chlamy does not take up more nitrate than untransformed control.
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</div></html>
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<html><div style="text-align: center; 20px;">
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<img src="https://static.igem.wiki/teams/5078/results/2-media-max-uptake-comparision.webp" width="600" height="auto"/><br>Figure 5. 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.
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</div></html>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 15:26, 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.

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 2. pL2-DeSlimer diagnostic digest using EcoRI on a 0.8% agarose gel. We selected colonies 6 and 9 for electropoation into chlamy.

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 3. These results show that our untransformed chlamy does not take up more phosphate than untransformed control.


Figure 4. These results show that our untransformed chlamy does not take up more nitrate than untransformed control.


Figure 5. 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]