Fig 1.
Identifying mNG variants with improved brightness in E. coli through directed evolution.
A. Schematics of mNG screening platform; B. Fluorescence images of E. coli colonies expressing split mNG2, mNG3A and mNG3K; C. Protein sequence alignment of full length mNG, mNG2, mNG3A and mNG3K. Chromophore amino acids are indicated by a red box and the split site is indicated by the small red arrow. The position of α-helices and β-sheets are shown and numbered above the alignment.
Fig 2.
Identifying split CloGFP by recovery brightness through directed evolution.
A. Schematics of split CloGFP screening platform; B. E. coli colony fluorescence intensity change during rounds of directed evolution, multiple colonies with same mutant were measured in R4; C. Protein sequence alignment of sfGFP, mClover3, split Clover0 and split CloGFP. Chromophore amino acids are indicated by a red box and the split site is indicated by the small red arrow. The position of α-helices and β-sheets are shown and numbered above the alignment.
Fig 3.
Improved affinity between FP1-10 and FP11 after engineering in mammalian cells.
A-D, flow cytometry analysis of cell fluorescence by transient transfection in HEK293T expressing A. full length mNG2, B. mNG21-10/11, C. mNG3A1-10/11, and D. mNG3K1-10/11. E-F flow cytometry analysis of cell fluorescence by transient transfection in HEK293T expressing E. CloGFP01-10/11 and F. CloGFP1-10/11. The data are normalized to a full-length control.
Fig 4.
Significant signal amplification by tandem mNG211 with mNG3A1-10 and mNG3K1-10.
A-C. Fluorescence intensity distribution of individual cells transiently co-transfected with FP11 (1x mNG211 in green or 5x mNG211 in pink) and FP1-10 (mNG21-10, mNG3A1-10 or mNG3K1-10), each condition has four replicates, solid lines show the mean of four distributions, the shadow indicates standard deviation. Representative images of the cells are shown in S4 and S5 Figs in S1 File. D. Each marker shows the median nuclear intensity for a field of view represented in A-C. Bars indicate the mean of all fields. Increased signal for mNG3A and mNG3K using 5x mNG211 is statistically significant (Welch’s t-test).