Development of a functional screen for FRET sensor performance in bacteria

With targeted mutagenesis of specific amino acids within Rsg-1.2 we were able to manipulate pH-dependency. With the introduction of linker sequences between the peptide and the FPs we aimed at decreasing the initial ratio and thereby increasing the dynamic range. However, one by one testing of several linkers showed unpredictable characteristics regarding initial ratio and ratio change. Consequently, a large number of random variants had to be created and tested following the model of molecular evolution. The generation of

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a huge number of candidate sensors necessitates the establishment of a suitable testing system for high-throughput screening. Bacteria colonies allow both testing in a biological context and easy handling of a large number of library members.

In order to develop a functional screen for improved sensor characteristics we tested FRET sensor performance in bacterial colonies. FRET ratios of bacterial colonies expressing FR-Rsg 7A8Q were significantly lower compared to colonies expressing both the sensor and 10 repeats of an aptamer at different time points after transformation (Figure 41A). In an inverse approach, we used a membrane to blot colonies expressing FR-Rsg1.2 and the aptamer repeats fused to mKO2 on a plate lacking one antibiotic necessary to select for the aptamer repeats. Lacking selection pressure, bacteria lose the mKO2-10xaptamer plasmid visualized by decreased mKO2 emission (Figure 41B). The decrease in FRET ratio after blotting was not significant. Nonetheless, both experiments show that the bacterial system is suitable for screening of FR-Rsg1.2 variants.

Figure 41 FR-Rsg1.2 7A8Q performance in bacteria. (A) Cotransformation of E. coli XL1 blue with FR- Rsg 7A8Q and 10xaptamer (red) compared to transformation with FR-Rsg 7A8Q only (grey). FRET ratio was recorded at different days after transformation. (B) Colonies from a bacterial plate transformed with FR-Rsg 7A8Q and an mKO2-10xaptamer construct (red filling) were subsequently transferred to a plate with no ampicillin using a Nitrocellulose membrane. Loss of selection pressure for ampicillin restisteny conferred by mKO2-10xaptamer leads to removal of this plasmid after 2

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blotting steps (grey filling). Average colony mKO2 emission (dark red stripes) and cpCitrine/eCFP ratio (black stripes) are plotted.

For the generation of a mutagenized sensor library we followed 3 different strategies (Figure 42A). In a random approach we performed error-prone PCR on the Rsg 7A8Q peptide using CFP Chr for and cpCit Chr rev primers thereby introducing mutations at undefined sites. Using another strategy, degenerated primers (Eurofins) allowed the introduction of randomized amino acids at defined positions during normal PCR reaction (see 3.2.1). The last library comprises linker constructs with random amino acids at both Rsg 7A8Q peptide- FP junctions. The peptide was amplified using degenerated primers that introduce triplets coding for 1, 2, 3, 4 or 8 amino acids. Resulting peptide elongation was supposed to conserve peptide dynamics while increasing the distance between the FRET pair.

The mutagenized peptide libraries were digested cloned into SphI/SacI and ligated into a pRSFDuet-eCFP-cpCit vector (Kana+) for weak protein expression. 10 aptamer repeats were cloned using HindIII/NotI into pMB133 which is designed to turn off the expression of the aptamer-containing RNA via TetR-tetO interactions in the promoter region (Valencia-Burton et al. 2009). This inhibition is released in the presence of anhydrotetracycline (aTC) which results in RNA aptamer expression. The sensor library was co-transformed into XL1 blue together with the pMBα3-10xRRE plasmid (Amp+).

On every bacterial plate, we placed two control patches with colonies expressing FR-Rsg 7A8Q only or FR-Rsg 7A8Q and pMB133-10xRRE. Using wide field CCD camera imaging of bacterial agar plates we were able to image simultaneously up to 1000 bacterial colonies each harboring a diversified sensor and to follow FRET changes upon induction of aptamer transcription (Figure 21) using 20 ng/µL aTC. For data analysis, one ROI per colony was automatically selected by a program written by David Ng using Cell Profiler. Data were analyzed by Origin 8.1 by plotting the ratio change 200 min after aTC induction over the starting FRET ratio. The selected colonies correspond to the ROIs with a ratio change higher and a starting ratio lower than the average of 6 control colonies.

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Figure 42 Screening for aptamer-induced FRET ratio change in bacteria. (A) Library generation by error prone PCR of Rsg1.2, mutagenesis at selected positions within the peptide or FP-peptide linkers of variable length and aa composition. (B) The sensor library is cotransformed with a 10xRRE construct controled by a tet promoter. Addition of aTc to the bacterial colonies on the plate induces 10xRRE transcription (red colonies). (C) FRET ratio is recorded before and after addition of aTC for 2- 4 h for all colonies from the library (grey) and the control colonies expressing FR-Rsg1.2 7A8Q with (red) and without the inducible aptamer (black). n=4 (D) A number is assigned to each colony (grey) and starting ratio is plotted against the relative temporal ratio change. 4 control conlonies expression FR-Rsg1.2 7A8Q with (red; VAmPIRe, orange) and without the inducible aptamer (black; VAmPIRe, blue). Variants with high ratio change and low starting ratios are picked and in vitro

tested. n=4

Overall about 60000 indicator variants with diversified linkers were pre-screened by imaging and 1500 indicators selected and subsequently purified for further spectroscopic analysis in vitro. Some variants with a starting ratio close to 1 showed lower ratio change after aptamer addition compared to FR-Rsg7A8Q (#47) (Figure 43). Evolutionary linker extension yielded variants with significantly enhanced FRET change, with the best variants (#678, # 95) now showing up to 160 % maximal change in emission ratio after aptamer binding (Figure 43). The best evolved sensor was called VAmPIRe (Viral Aptamer binding Peptide based Indicator for RNA detection).

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Figure 43 Performance of selected sensor variants. (A) Ratio change is plotted for FR-Rsg1.2 (wt, grey), FR-Rsg1.2 7A8Q (red) and sensor variants selected by bacterial screening (blue, green). (B)

Peptide sequences of the variants in (A).