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Part:BBa_K4201021:Design
CrtE_cytoTDS2-MBP_T5αOH_RUBY
- 10INCOMPATIBLE WITH RFC[10]Unknown
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25INCOMPATIBLE WITH RFC[25]Unknown
- 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 3114
Illegal BsaI site found at 6693
Illegal BsaI site found at 6973
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 6679
Illegal BsaI.rc site found at 6959
Illegal BsaI.rc site found at 7906
Design Notes
Part information and design consideration can be found on respective basic parts pages.
This construct differs from similar composite parts like BBa_K4201019 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.
RUBY, which is added to the end of this assembly, is a reporter gene that signifies if gene transformation was successful in Glycine max4.
Compared to BBa_K4201020, this construct contains the coding sequence for a maltose binding protein (MBP), which helps maintain protein stability during transport5.
Gmubi is constitutive promoter native to Glyine max6, while AtHSP is the terminator of a heat shock protein that has shown to promote expression in plants7.
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 is a GGPP synthase from Pantoea ananatis LMG 20103
RUBY is a reporter gene from the order Caryophyllales.
Gmubi promoters originate from Glycine max.
AtHSP terminators originate from the Arabidopsis thaliana genome.
cytoTDS2 is a taxadiene synthase native to Taxus chinensis var. mairei optimized for use in Glycine max.
T5αOH is a hydroxylase from Taxus baccata that converts taxadiene into taxadiene-5α-ol, a step in the paclitaxel pathway.
de novo Synthesis was completed by iGem sponsors IDT and Twist Biosciences.