Part:BBa_K3726016:Design
CDS_Lv0_BOH_2_B
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1246
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 252
Illegal NheI site found at 480
Illegal PstI site found at 1246 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1591
Illegal BglII site found at 1708 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1246
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1246
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
In the upstream region the bases taatc corresponds with 5bp a spacer sequence to improve ribosomal translation, while AATG overhang creates an start codon that forces translation initiation of the downstream coding sequence.
In the downstream region, the extra “aa” bases allow the creation of an additional stop codon (TAA) at the end of the upstream coding sequence, ending the translation of the desired CDS in accordance with the Marburg Collection design guidelines.
The transcription initiation rate for each RBS element has been specifically adjusted to achieve optimal expression of the desired enzyme within the operon. The relative translation initiation rates for the operon can be observed in the picture below. Where this operon corresponds with the enzymes: CDS_PduP and CDS_Slr1192.
This polycistronic CDS sequence has been assembled following a modified procedure for golden gate domestication. To know more about the design process of this polycistronic lv.0 parts, visit the iGEM MADRID_UCM 2021 wiki page https://2021.igem.org/Team:MADRID_UCM/Design.
Source
Firstly we made two PCR domestication with specific primers and CDS_LV0_BOH_3 ("BBa_K3726011") as template obtaining as products two sequences that afterwards were linked with Golden Gate. That sequences were able to be attached by golden gate because they had few complementary nucleotide as overhangs.
References
X. Liu, R. Miao, P. Lindberg and P. Lindblad, "Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2in cyanobacteria", 2021.