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. 2017 Nov 13;6:e28921. doi: 10.7554/eLife.28921

Figure 6. Not all intermolecular epistasis can be explained by accounting for the underlying genetic regulatory structure of the system.

We created 150 double mutants with single mutation combinations corresponding to Figure 5G. A double mutant in this library would not be in epistasis unless a mutation in trans binds the cis mutant differently than the wildtype trans does. In a plate reader, we measured expression levels of monoclonal populations of each double mutant and its constitutive single mutants in the presence of CI. Epistasis was estimated as the deviation of the observed double mutant effect from the additive expectation based on single mutant effects. The grey bar indicates measurements that are not in significant epistasis. The effects of single mutations in cis and in trans, as well as the double mutant effects, are shown in Figure 6—figure supplement 1, while the underlying data are shown in Figure 6—source data 1. The location of point mutations is shown in Figure 6—figure supplement 2 and Figure 6—source data 2.

Figure 6—source data 1. Calculating epistasis from the effects of 150 double mutants and their corresponding single point mutations, measured in plate reader.
This library consists only of point mutants in cis that lead to high expression in the absence of CI, and point mutants in trans that result in no expression in the presence of CI. The data provided here are shown in Figure 6, and Figure 6—figure supplement 1.
elife-28921-fig6-data1.xlsx (68KB, xlsx)
DOI: 10.7554/eLife.28921.033
Figure 6—source data 2. Identity and location of mutations in the library of 150 double mutant, with a point mutation in cis that leads to high expression in the absence of CI, and a point mutation in trans that leads to no expression in the presence of CI.
elife-28921-fig6-data2.xlsx (52.5KB, xlsx)
DOI: 10.7554/eLife.28921.034

Figure 6.

Figure 6—figure supplement 1. Distribution of single mutation effects in 150 system double mutants and their corresponding single mutants.

Figure 6—figure supplement 1.

We created 150 unique double mutants, with one point mutation in cis that had high expression in the absence of CI, and the other in trans that exhibited no expression in the presence of CI. We measured gene expression of each mutant at a population level in a plate reader. Histograms of expression levels in the absence and in the presence of CI are shown for: (A) point mutations in cis; (B) point mutations in trans; (C) double mutants in the system. Dotted line represents mean wildtype fluorescence in the corresponding environment. Six replicates of each mutant were measured. Grey bars indicate mutants that were not significantly different from the wildtype. The data underlying this figure are shown in Figure 6—source data 1.
Figure 6—figure supplement 2. Identity and location of mutations in the double mutant library with a trans mutation that has no expression in the presence of CI, and a cis mutation with high expression in the absence of CI.

Figure 6—figure supplement 2.

(A) DNA sequence of the trans-element showing the N-terminal domain (red), C-terminal domain (blue), and the linker region connecting the two domains (black). (B) DNA sequence of the cis-element showing the −35 and −10 RNAP recognition sites (from left to right, underlined), and the two CI repressor operators (green). Unlike the double mutant library shown in Figure 3, where each double mutant had a unique mutation in cis and trans, in this library some cis mutations had to be repeated, as we could not identify 150 mutations in cis that had high expression in the absence of CI.