Part:BBa_K2805004:Design

LOV2 -> mRuby

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal suffix found in sequence at 1086
  Illegal BglII site found at 369
  Illegal BglII site found at 2342
  Illegal BamHI site found at 126
  Illegal BamHI site found at 3239
  Illegal XhoI site found at 3074
  Illegal XhoI site found at 3248
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1406
  Illegal NgoMIV site found at 2644
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part is the LOV2 promoter driving expression of mRuby, a composite of BBa_K2805002 and BBa_K2805003. An organism expressing BBa_K2805004 will produce mRuby, a red fluorescent protein, when under a blue light (450-490 nm). This sequence is optimized for integration into S. cerevisiae

The primary concern was Dueber MoClo and GoldenGate compatibility. These sequences were designed to be robustly assembled and integrated into the S. cerevisiae genome.

Source

LOV2 was identified and engineered from E. litoralis by Laura B. Motta-Mena et al. (Nature Chemical Biology 2014, doi: 10.1038/nchembio.1430). The C120op EL222-binding sequence was first identified in E. litoralis by Giomar Rivera-Cancel, Laura B. Motta-Mena, and Kevin H. Gardner (Biochemistry 2012, doi: 10.1021/bi301306t). mRuby2 is derived from Entacmaea quadricolor, best described by Aj et al. (Nature Methods 2012, doi: 10.1038/nmeth.2171).

References

1. Benzinger D, Khammash M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nature Communications. 2018;9(1):3521. https://doi.org/10.1038/s41467-018-05882-2. doi: 10.1038/s41467-018-05882-2.

2. Motta-Mena L, Reade A, Mallory MJ, et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature Chemical Biology. 2014;10:196. http://dx.doi.org/10.1038/nchembio.1430.

3. Zhao EM, Zhang Y, Mehl J, et al. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature. 2018;555:683. http://dx.doi.org/10.1038/nature26141.

4. Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol. 2015;4(9):975-986. https://doi.org/10.1021/sb500366v. doi: 10.1021/sb500366v.