Device

Part:BBa_M50560:Design

Designed by: Maria Paula Hernandez   Group: Stanford BIOE44 - S11   (2018-12-12)


engineered E.coli with blue light-dependent switch


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE 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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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