Part:BBa_K4201016:Design
Crte-cytoTDS-MBP_RUBY
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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).