Part:BBa_K4201017:Design
CrtE_cytoTDS2_RUBY
- 10INCOMPATIBLE WITH RFC[10]Unknown
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 790
Illegal BglII site found at 1153
Illegal BglII site found at 2021
Illegal BglII site found at 2956
Illegal BglII site found at 4011
Illegal BglII site found at 5324
Illegal BglII site found at 5572
Illegal BamHI site found at 9058
Illegal XhoI site found at 3601
Illegal XhoI site found at 3868 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 2087
Illegal PstI site found at 5638
Illegal PstI site found at 10846 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 2087
Illegal PstI site found at 5638
Illegal PstI site found at 10846
Illegal NgoMIV site found at 1697
Illegal NgoMIV site found at 6994
Illegal NgoMIV site found at 7573
Illegal NgoMIV site found at 9286 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI site found at 948
Illegal BsaI site found at 1887
Illegal BsaI site found at 2167
Illegal BsaI site found at 3113
Illegal BsaI site found at 5438
Illegal BsaI site found at 5718
Illegal BsaI.rc site found at 934
Illegal BsaI.rc site found at 1873
Illegal BsaI.rc site found at 2153
Illegal BsaI.rc site found at 3100
Illegal BsaI.rc site found at 5424
Illegal BsaI.rc site found at 5704
Illegal BsaI.rc site found at 6651
Design Notes
Part information and design consideration can be found on respective basic parts pages.
This construct differs from similar composite parts like BBa_K4201016 and 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 max4, while AtHSP is the terminator of a heat shock protein that has shown to promote expression in plants5.
This sequence, unlike our other constructs notably does not contain the sequence encoding Maltose-binding protein (MBP), which was done purposely to determine if MBP had an impact on taxadiene production in Glycine max
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
Gmubi promoters originate from Glycine max4.
AtHSP terminators originate from the Arabidopsis thaliana genome5.
CrtE is a GGPP synthase from Pantoea ananatis LMG 201031.
cytoTDS2 is a taxadiene synthase native to Taxus chinensis var. mairei optimized for use in Glycine max2..
RUBY is a reporter gene from the order Caryophyllales3.
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. 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).
4. 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).
5. 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).