Part:BBa_K2680255

3G mScarlet-I pdt#3d

3G mScarlet-I with pdt#3d degradation tag.

Description and Characterization

Coding Sequence for red fluorescent protein mScarlet-I followed by pdt#3d degradation tag (medium weak degradation tag). Compatible with Team William_and_Mary iGEM 2018's 3G format as well as CIDAR MoClo assembly. mScarlet-I is the rapidly-maturing version of mScarlet, a monomeric red fluorescent protein designed by Bindels et al. in 2016. mScarlet-I is distinguished from the original mScarlet protein by a single amino acid substitution (T74I). mScarlet-I has a peak excitation at 569nm and peak emission at 594nm, and possesses both a high quantum yield (0.54) and fluorescence lifetime (3.1ns), and a rapid maturation time of ~36 minutes [1].

3G mScarlet-I characterized against variants of 3G mScarlet-I with pdt degradation tags. 3G mScarlet-I serves as a control—without a pdt tag, 3G mScarlet-I will not be degraded by mf-lon. 3G mScarlet-I's prominent fluorescence matched expectations based on its rapid maturation rate. mf-lon will degrade 3G mScarlet-I when the part is followed by a pdt degradation tag. Of pdt#3 through pdt#3e, pdt#3 is the strongest degradation tag and pdt#3e is the weakest. Following the decrease in strength from pdt#3 to pdt#3e, pdt#3d is a medium-weak degradation tag.

Improvement

3G mScarlet-I is flanked by prefix sticky end C and suffix sticky end D in a 3G-compatible backbone. These additions allow for the use of 3G mScarlet-I in 3G Assembly, a Gibson-Golden Gate hybrid assembly method. Specifically, 3G mScarlet-I's sticky ends correspond to the suffix sticky end C in all 3G RBS and the prefix sticky end D in all 3G terminators. This allows for great modularity in the Golden Gate reaction of 3G, as various 3G parts can be combined with 3G mScarlet-I to produce unique transcriptional units. 3G's modularity continues into the following Gibson reaction, where transcriptional units with corresponding UNS sequences can be "swapped out" to create unique circuits.

As 3G mScarlet-I is 3G-compatible, and thus available for efficient and modular assembly with other 3G parts, it represents an improvement from the previous mScarlet-I.

References

[1] Bindels, D. S., Haarbosch, L., Weeren, L. V., Postma, M., Wiese, K. E., Mastop, M. Gadella, T. W. (2016). MScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods, 14(1), 53-56. doi:10.1038/nmeth.4074

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 560
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 41
  Illegal BsaI.rc site found at 837