Translational_Unit
OsPHT1;6 T

Part:BBa_K4213040

Designed by: Eleftheria Lakaki   Group: iGEM22_Thessaly   (2022-09-04)


TetO7-pTriple-OsPHT1;6(dom&opt):Venus:tNOS(stuffer)

Engineering Cycle 3: Second Iteration: Establishing Transporter Expression

Design

After the confirmation of expression, we could move on to our phosphorus transporter. We decided to test the genes of PHT1;5 from Arabidopsis thaliana [1] [2] and PHT1;6 from Oryza sativa [3] [4] Pi transporters due to their evolutionary relatedness of each source organism to our end-product plant (P. australis) and their potential to be overexpressed without showing significant levels of toxicity, leading to even higher Pi uptake regardless of its concentration [5]. So after finding the cds of each gene, we did two rounds of modifications. Firstly, we searched for the same Kozak Frame motif around the TIS, leading to two substitutions in the OsPHT1;6 sequence (+5: G>C, +6: A>T) and a mutation in the aa sequence (G2A) which should not cause a problem to the functionality of the transporter due to the size and the place of the two aa. Secondly, we decided to test the effects of codon optimization on these transporters by ordering both codon optimized and not. Sadly, the high complexity of the OsPHT1;6 sequence did not allow us to order it from neither IDT nor TWIST, therefore we partially optimized the spots characterized as “highly complex” by the tool. Finally, as again we planned to visualize expression through fluorescent microscopy, we included a linker sequence and our reference gene (mVenus) downstream of the part. To summarize, our final versions of construct 2 were:


  1. mod-p35s:(optimized)AtPHT1;5:Venus:tNOS (BBa_K4213035)
  2. mod-p35s:(non-optimized)AtPHT1;5:Venus:tNOS (BBa_K4213036)
  3. mod-p35s:(optimized)OsPHT1;6:Venus:tNOS (BBa_K4213040)
  4. mod-p35s:(partially-optimized)OsPHT1;6:Venus:tNOS (BBa_K4213043)

Build

Repeating the same steps and after several weeks of cloning, together with sequencing, we managed to confirm the assembly of the abovementioned constructs.

Diagnostic_Digestion
Figure 1: Diagnostic Digestion of:
  1. 2 and 3 “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 1492, 674 and 387)
  2. 4 and 5 “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 908, 674, 584 and 387)
  3. 6 and 7 “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 2041, 1405, 674 and 387)
  4. 8 and 9 “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 3449, 674 and 387)

Positive results: 2, 4 and 5 MW: 1

Test

Again, after agroinfiltration and preparation of samples like before, we tried to observe our constructs under the confocal microscope.

Learn

Unfortunately, we encountered a problem with the setup of our confocal microscope and due to the time constraints we were not able to rerun the experiment in order to confirm the expression and localization of the transporters.

References

[1] Smith, A. P., Nagarajan, V. K., & Raghothama, K. G. (2011b, November). Arabidopsis Pht1;5 plays an integral role in phosphate homeostasis. Plant Signaling &Amp; Behavior, 6(11), 1676–1678. https://doi.org/10.4161/psb.6.11.17906

[2] Nagarajan, V. K., Jain, A., Poling, M. D., Lewis, A. J., Raghothama, K. G., & Smith, A. P. (2011b, May 31). Arabidopsis Pht1;5 Mobilizes Phosphate between Source and Sink Organs and Influences the Interaction between Phosphate Homeostasis and Ethylene Signaling. Plant Physiology, 156(3), 1149–1163. https://doi.org/10.1104/pp.111.174805

[3] Ai, P., Sun, S., Zhao, J., Fan, X., Xin, W., Guo, Q., Yu, L., Shen, Q., Wu, P., Miller, A. J., & Xu, G. (2009b, March). Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. The Plant Journal, 57(5), 798–809. https://doi.org/10.1111/j.1365-313x.2008.03726.x

[4] Zhang, F., Wu, X. N., Zhou, H. M., Wang, D. F., Jiang, T. T., Sun, Y. F., Cao, Y., Pei, W. X., Sun, S. B., & Xu, G. H. (2014b, July 25). Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants. Plant and Soil, 384(1–2), 259–270. https://doi.org/10.1007/s11104-014-2168-8

[5] Gu, M., Chen, A., Sun, S., & Xu, G. (2016b, March). Complex Regulation of Plant Phosphate Transporters and the Gap between Molecular Mechanisms and Practical Application: What Is Missing? Molecular Plant, 9(3), 396–416. https://doi.org/10.1016/j.molp.2015.12.012

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 353
    Illegal PstI site found at 347
    Illegal PstI site found at 2023
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 353
    Illegal NheI site found at 2088
    Illegal PstI site found at 347
    Illegal PstI site found at 2023
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 353
    Illegal BamHI site found at 2497
    Illegal XhoI site found at 34
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 353
    Illegal PstI site found at 347
    Illegal PstI site found at 2023
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 353
    Illegal PstI site found at 347
    Illegal PstI site found at 2023
    Illegal NgoMIV site found at 28
  • 1000
    COMPATIBLE WITH RFC[1000]


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