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Large Scale Bacterial Colony Screening of Diversified FRET Biosensors.

Litzlbauer J, Schifferer M, Ng D, Fabritius A, Thestrup T, Griesbeck O - PLoS ONE (2015)

Bottom Line: Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry.Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors.We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.

ABSTRACT
Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors.

No MeSH data available.


Related in: MedlinePlus

Optimization of expression conditions and screening background.(A) Colonies of E. coli BL21 transformed with TN-XXL cloned into the vector pRSETB (exposure time 4s, gain 2x, binning 1). Scale bar, 10 mm (B) Colonies of E. coli XL1 transformed with TN-XXL cloned into pRSETB and imaged under the same conditions. (C) Mean fluorescence intensities ± SD of colonies transformed with TN-XXL in either BL1 or XL1, imaged under identical conditions (nBL21 = 312, nXL1 = 558). (D) ΔR/R ± SD of E. coli colonies expressing TN-XXL cloned into BL21 or XL1 (nBL21 = 19, nXL1 = 15). (E) Auto-fluorescence of agar plate versus white blotting paper imaged under identical conditions with filters used for FRET imaging. Error bars indicate SD. (F) Basal ratio valuesR0 ± SD of XL1 colonies transformed with the FRET sensor TN-XXL and imaged on agar plate or on filter paper. (nplate = 149 colonies, nblotting paper = 90 colonies) (G) ΔR/R ± SD of the same colonies following calcium application.
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pone.0119860.g002: Optimization of expression conditions and screening background.(A) Colonies of E. coli BL21 transformed with TN-XXL cloned into the vector pRSETB (exposure time 4s, gain 2x, binning 1). Scale bar, 10 mm (B) Colonies of E. coli XL1 transformed with TN-XXL cloned into pRSETB and imaged under the same conditions. (C) Mean fluorescence intensities ± SD of colonies transformed with TN-XXL in either BL1 or XL1, imaged under identical conditions (nBL21 = 312, nXL1 = 558). (D) ΔR/R ± SD of E. coli colonies expressing TN-XXL cloned into BL21 or XL1 (nBL21 = 19, nXL1 = 15). (E) Auto-fluorescence of agar plate versus white blotting paper imaged under identical conditions with filters used for FRET imaging. Error bars indicate SD. (F) Basal ratio valuesR0 ± SD of XL1 colonies transformed with the FRET sensor TN-XXL and imaged on agar plate or on filter paper. (nplate = 149 colonies, nblotting paper = 90 colonies) (G) ΔR/R ± SD of the same colonies following calcium application.

Mentions: The goal was to select sensors with large FRET changes out of a library of diversified sensors that would encompass a broad range of FRET ratio values both under resting conditions and in ligand bound state. These sensor libraries were transformed into bacteria and the bacteria were plated onto LB agar plates. We used a standard filter wheel set-up and wide-field imaging with a CCD camera to monitor the bacterial colonies expressing fluorescent biosensors (Fig 1). With this approach it could be ensured that a large number of sensor variants could be sampled simultaneously. Then we set out to optimize the bacterial strains used, expression and imaging conditions, permeabilization protocols to measure Rminin ligand free and Rmaxin ligand-bound state of the sensors, and software routines to automate colony identification and analysis of responses. We initially sought to find suitable conditions to express and image biosensors in E coli. For this purpose we used TN-XXL, a well described FRET-based calcium sensor [24,25], cloned into the bacterial expression plasmid pRSETB (Invitrogen), to test various bacterial strains and conditions. We compared sensor response in a bacterial strain used for protein expression, BL21-Gold (Stratagene), and a strain used for cloning, XL1-Blue (Stratagene) (Fig 2). As expected, BL21-Gold displayed a fluorescence intensity more than 2 times higher than that of XL1-Blue when transformed with vectors coding for TN-XXL (Fig 2A–2C).However, we typically observed that FRET changes, e.g. after permeabilization of the bacterial colonies for calcium, were consistently higher in XL1-Blue (Fig 2D). Lower, leaky expression of the biosensor in the strain XL1-Blue, which was intended not to express any recombinant proteins from transformed plasmids, avoided dense packing artefacts and other potential detrimental effects of protein overexpression in the bacterial cytosol. As the brightness in XL1-Blue still provided enough photons for screening we chose this strain for further work. Bacterial plates were transformed with biosensors and incubated at 37°C for about 16–18 hours. After that time, colonies were large enough to be used, yet no non-resistant satellite colonies were visible surrounding the main colonies. Subsequently, the plates were left at room temperature for another 5–6 hours to allow the proteins to further maturate, and then stored at 4°C overnight, before they were imaged on the second day. Colonies were blotted from agar plates onto blotting paper (Whatman 3MM) presoaked in MOPS buffer before imaging. This measure was thought to be beneficial since the agar plates exhibited autofluorescence which was reduced by white paper background (Fig 2E). Starting ratios (R0) of TN-XXL on plate and blotting paper were similar (Fig 2F). When treated with permeabilizing agents and high calcium however, the ΔR/R of TN-XXL was 4 times higher in colonies imaged on paper in comparison to those imaged on plates (Fig 2G). Presumably the blotting paper is more suitable to distribute the solution for permeabilization around the colonies to allow better penetration into the colonies. The blotting paper containing the colonies was then placed on a movable stage and imaged using the CCD camera set-up at room temperature (Fig 1).


Large Scale Bacterial Colony Screening of Diversified FRET Biosensors.

Litzlbauer J, Schifferer M, Ng D, Fabritius A, Thestrup T, Griesbeck O - PLoS ONE (2015)

Optimization of expression conditions and screening background.(A) Colonies of E. coli BL21 transformed with TN-XXL cloned into the vector pRSETB (exposure time 4s, gain 2x, binning 1). Scale bar, 10 mm (B) Colonies of E. coli XL1 transformed with TN-XXL cloned into pRSETB and imaged under the same conditions. (C) Mean fluorescence intensities ± SD of colonies transformed with TN-XXL in either BL1 or XL1, imaged under identical conditions (nBL21 = 312, nXL1 = 558). (D) ΔR/R ± SD of E. coli colonies expressing TN-XXL cloned into BL21 or XL1 (nBL21 = 19, nXL1 = 15). (E) Auto-fluorescence of agar plate versus white blotting paper imaged under identical conditions with filters used for FRET imaging. Error bars indicate SD. (F) Basal ratio valuesR0 ± SD of XL1 colonies transformed with the FRET sensor TN-XXL and imaged on agar plate or on filter paper. (nplate = 149 colonies, nblotting paper = 90 colonies) (G) ΔR/R ± SD of the same colonies following calcium application.
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pone.0119860.g002: Optimization of expression conditions and screening background.(A) Colonies of E. coli BL21 transformed with TN-XXL cloned into the vector pRSETB (exposure time 4s, gain 2x, binning 1). Scale bar, 10 mm (B) Colonies of E. coli XL1 transformed with TN-XXL cloned into pRSETB and imaged under the same conditions. (C) Mean fluorescence intensities ± SD of colonies transformed with TN-XXL in either BL1 or XL1, imaged under identical conditions (nBL21 = 312, nXL1 = 558). (D) ΔR/R ± SD of E. coli colonies expressing TN-XXL cloned into BL21 or XL1 (nBL21 = 19, nXL1 = 15). (E) Auto-fluorescence of agar plate versus white blotting paper imaged under identical conditions with filters used for FRET imaging. Error bars indicate SD. (F) Basal ratio valuesR0 ± SD of XL1 colonies transformed with the FRET sensor TN-XXL and imaged on agar plate or on filter paper. (nplate = 149 colonies, nblotting paper = 90 colonies) (G) ΔR/R ± SD of the same colonies following calcium application.
Mentions: The goal was to select sensors with large FRET changes out of a library of diversified sensors that would encompass a broad range of FRET ratio values both under resting conditions and in ligand bound state. These sensor libraries were transformed into bacteria and the bacteria were plated onto LB agar plates. We used a standard filter wheel set-up and wide-field imaging with a CCD camera to monitor the bacterial colonies expressing fluorescent biosensors (Fig 1). With this approach it could be ensured that a large number of sensor variants could be sampled simultaneously. Then we set out to optimize the bacterial strains used, expression and imaging conditions, permeabilization protocols to measure Rminin ligand free and Rmaxin ligand-bound state of the sensors, and software routines to automate colony identification and analysis of responses. We initially sought to find suitable conditions to express and image biosensors in E coli. For this purpose we used TN-XXL, a well described FRET-based calcium sensor [24,25], cloned into the bacterial expression plasmid pRSETB (Invitrogen), to test various bacterial strains and conditions. We compared sensor response in a bacterial strain used for protein expression, BL21-Gold (Stratagene), and a strain used for cloning, XL1-Blue (Stratagene) (Fig 2). As expected, BL21-Gold displayed a fluorescence intensity more than 2 times higher than that of XL1-Blue when transformed with vectors coding for TN-XXL (Fig 2A–2C).However, we typically observed that FRET changes, e.g. after permeabilization of the bacterial colonies for calcium, were consistently higher in XL1-Blue (Fig 2D). Lower, leaky expression of the biosensor in the strain XL1-Blue, which was intended not to express any recombinant proteins from transformed plasmids, avoided dense packing artefacts and other potential detrimental effects of protein overexpression in the bacterial cytosol. As the brightness in XL1-Blue still provided enough photons for screening we chose this strain for further work. Bacterial plates were transformed with biosensors and incubated at 37°C for about 16–18 hours. After that time, colonies were large enough to be used, yet no non-resistant satellite colonies were visible surrounding the main colonies. Subsequently, the plates were left at room temperature for another 5–6 hours to allow the proteins to further maturate, and then stored at 4°C overnight, before they were imaged on the second day. Colonies were blotted from agar plates onto blotting paper (Whatman 3MM) presoaked in MOPS buffer before imaging. This measure was thought to be beneficial since the agar plates exhibited autofluorescence which was reduced by white paper background (Fig 2E). Starting ratios (R0) of TN-XXL on plate and blotting paper were similar (Fig 2F). When treated with permeabilizing agents and high calcium however, the ΔR/R of TN-XXL was 4 times higher in colonies imaged on paper in comparison to those imaged on plates (Fig 2G). Presumably the blotting paper is more suitable to distribute the solution for permeabilization around the colonies to allow better penetration into the colonies. The blotting paper containing the colonies was then placed on a movable stage and imaged using the CCD camera set-up at room temperature (Fig 1).

Bottom Line: Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry.Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors.We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.

ABSTRACT
Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors.

No MeSH data available.


Related in: MedlinePlus