Part:BBa_M50560:Design
engineered E.coli with blue light-dependent switch
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 691
Illegal NheI site found at 1746
Illegal NheI site found at 1809
Illegal NheI site found at 1832
Illegal NheI site found at 3003 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 141
Illegal NgoMIV site found at 434
Illegal NgoMIV site found at 528
Illegal NgoMIV site found at 660
Illegal NgoMIV site found at 2408
Illegal NgoMIV site found at 2480
Illegal NgoMIV site found at 2570
Illegal AgeI site found at 309
Illegal AgeI site found at 2122 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 198
Illegal BsaI.rc site found at 2021
Design Notes
Plasmid 1: KMB_pColi-Blue Light System (YF1) FINAL
For our device, we need the blue light sensor to always be present, so we chose a constitutively active promoter (BBa_J23108) for the expression of YF1, the protein that actually interacts with blue light. Again, since we want strong expression, we chose a strong RBS (BBa_M3771) to maximize the translation of the mRNA. The YF1 gene sequence (BBa_K592004) follows the RBS and codes for the expression of the YF1 protein. Transcription is terminated by the pT-T7 terminator (BBa_M36305). The plasmid also contains a the high copy number replication origin pUC, to ensure that the plasmid is produced and passed down to daughter cells, and an ampicillin resistance gene to select for successful transformation.
Plasmid 2: KMB_pColi-Blue Light System (FixJ) FINAL
The second half of the blue light sensor comes from the protein FixJ, which is phosphorylated by YF15. Since the output of this protein needs to approximately match that of the YF1 protein, we are using the same constitutively active promoter, strong RBS, and terminator. The FixJ gene (BBa_K592005) is placed appropriately between the RBS and terminator. Although putting FixJ on the same transcriptional unit as YF1 would also ensure matched expression, we did not do so because the resulting plasmid would be too long and would not be able to be easily cloned using Gibson assembly cloning. We would like for our device to output the fluorescent reporter protein mCherry in the absence of blue light. To regulate transcription, we chose the pFixK2 promoter (BBa_K592006), which promotes transcription when FixJ is phosphorylated11 (i.e. when there is no blue light). This is followed by the same strong RBS that was used before, then the mCherry gene (BBa_J06504), and ends with a pT-T7 terminator.
Source
https://parts.igem.org/Part:BBa_M50502- FixJ plasmid
https://parts.igem.org/Part:BBa_M50501- YF1 plasmid refer to: Fernandez-Rodriguez, et al. C. A. 2017. Engineering RGB color vision into Escherichia coli. Nature Chemical Biology, 13(7), 706-708. doi:10.1038/nchembio.2390
References
Fernandez-Rodriguez, et al. C. A. 2017. Engineering RGB color vision into Escherichia coli. Nature Chemical Biology, 13(7), 706-708. doi:10.1038/nchembio.2390