Composite

Part:BBa_K4201016:Design

Designed by: David Birkhaeuser   Group: iGEM22_CU-Boulder   (2022-10-12)


Crte-cytoTDS-MBP_RUBY


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1002
    Illegal PstI site found at 4800
    Illegal PstI site found at 5648
    Illegal PstI site found at 10856
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1002
    Illegal PstI site found at 4800
    Illegal PstI site found at 5648
    Illegal PstI site found at 10856
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 790
    Illegal BglII site found at 1618
    Illegal BglII site found at 3156
    Illegal BglII site found at 4203
    Illegal BglII site found at 5582
    Illegal BglII site found at 6517
    Illegal BglII site found at 10419
    Illegal BamHI site found at 5254
    Illegal BamHI site found at 9068
    Illegal XhoI site found at 2579
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1002
    Illegal PstI site found at 4800
    Illegal PstI site found at 5648
    Illegal PstI site found at 10856
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1002
    Illegal PstI site found at 4800
    Illegal PstI site found at 5648
    Illegal PstI site found at 10856
    Illegal NgoMIV site found at 7004
    Illegal NgoMIV site found at 7583
    Illegal NgoMIV site found at 9296
    Illegal AgeI site found at 2321
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI site found at 948
    Illegal BsaI site found at 5448
    Illegal BsaI site found at 5728
    Illegal BsaI site found at 6675
    Illegal BsaI site found at 10656
    Illegal BsaI.rc site found at 934
    Illegal BsaI.rc site found at 5434
    Illegal BsaI.rc site found at 5714
    Illegal BsaI.rc site found at 6661
    Illegal BsaI.rc site found at 10642
    Illegal BsaI.rc site found at 10922
    Illegal SapI.rc site found at 3377
    Illegal SapI.rc site found at 3583


Design Notes

Part information and design consideration can be found on respective basic parts pages.

This construct differs from similar composite parts like BBa_K4201018 by having different promoter (Gmubi) and terminator (AtHSP) amounts and locations. In these constructs, dashes(-) represent locations where genes were synthesized de novo. An underscore (_) symbolizes a location where we adhered genes together via Golden Gate assembly. Utilizing Golden Gate assembly allowed us to insert promoter and terminator sequences between genes of interest, and consequently enables the study of transcription efficacy based on promoter and terminator location.

Gmubi is constitutive promoter native to Glyine max5, while AtHSP is the terminator of a heat shock protein that has shown to promote expression in plants6.

This assembly was made using NEB 10-beta cells and GoldBio EHA105 agrobacterium as intermediate hosts. Parts BBa_K4201013 (Amp BsaI) and BBa_K4201014 (Chlor AaRI) were used as intermediate backbones for Golden Gate assembly. The construct was integrated into part BBa_K4201015 (Kan BsaI) as a final backbone before transfection into Glycine max.

Source

Crte-cytoTDS-MBP is a de novo synthesized construct made for this project. Learn more about our construct and its genes by searching for BBa_K4201004.

RUBY is a reporter gene from the order Caryophyllales4.

Gmubi promoters originate from Glycine max5.

AtHSP terminators originate from the Arabidopsis thaliana genome6.


Synthesis was completed by iGem sponsors IDT and Twist Biosciences.

References

1. Majer, E., Llorente, B., Rodríguez-Concepción, M. & Daròs, J.-A. Rewiring carotenoid biosynthesis in plants using a viral vector. Sci. Rep. 7, 41645 (2017).

2. Xiong, X. et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nat. Plants 7, 1026–1036 (2021).

3. Lebendiker, M. & Danieli, T. Purification of Proteins Fused to Maltose-Binding Protein. Methods Mol. Biol. Clifton NJ 1485, 257–273 (2017).

4. He, Y., Zhang, T., Sun, H., Zhan, H. & Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic. Res. 7, 1–6 (2020).

5. Lebendiker, M. & Danieli, T. Purification of Proteins Fused to Maltose-Binding Protein. Methods Mol. Biol. Clifton NJ 1485, 257–273 (2017).

6. De La Torre, C. M. & Finer, J. J. The intron and 5’ distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues. Plant Cell Rep. 34, 111–120 (2015).

7. Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP Terminator of Arabidopsis thaliana Increases Gene Expression in Plant Cells. Plant Cell Physiol. 51, 328–32 (2010).