Team:William and Mary/Improve


For William and Mary’s improved part, we modified mScarlet-I to be compatible with our 3G assembly method. mScarlet-I is a rapidly-maturing version of mScarlet, a red fluorescent protein designed by Bindels et al [1]. mScarlet-I differs from the original mScarlet protein by a single amino acid substitution T741.
We improved mScarlet-I by flanking it with 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. This 3G compatibility makes 3G mScarlet-I more modular and more efficient for DNA assembly than the previous mScarlet-I.Please see BBa_K2680251 for more characterization showing is an improved version of BBa_K2333009.
Figure 1: An example of transcriptional units created using 3G mScarlet-I. A is R0051 B0034 mScarlet-I Spy Terminator, with 959 base pairs. B is ptlpA B0034m mScarlet-I thru terminator, with 932 base pairs.
The gel extraction image above displays two transcriptional units, both containing mScarlet-I. Without the modifications from our sticky ends, these transcriptional units would not have been possible and it would have taken several times as long to assemble all the requisite parts needed for our circuit.
[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