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Tunable protein degradation in bacteria.

Cameron DE, Collins JJ - Nat. Biotechnol. (2014)

Bottom Line: Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli.We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria.We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Boston University, Boston, Massachusetts, USA. [2] Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA. [3] Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

ABSTRACT
Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.

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Pdt system characterization(a) Comparative analysis of pdt-mediated degradation of mCherry and GFP. Pdt letter variants were fused to GFP and mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (50 ng/ml ATc for 6 h). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=1.09× – 0.01 with an R2 value of 0.98 and standard errors of 0.03 and 0.01 for the slope and y-intercept, respectively. (b) Pdt-dependent degradation of mCherry in L. lactis. Nisin induced mf-Lon expression in L. lactis causes pdt-dependent mCherry degradation. Data show the geometric mean fluorescence as a percent of the fluorescence of uninduced cells. Nisin induction was 3 ng/ml. (c) Comparative analysis of pdt letter variants in E. coli and L. lactis. Pdt letter variants were fused to mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (6 hour induction, E. coli: 50 ng/ml ATc and L. lactis: 3 ng/ml nisin). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=0.51× – 0.04 with an R2 value of 0.91 and standard errors of 0.03 and 0.02 for the slope and y-intercept, respectively. (d) Transcription and post-translation-based control of mf-Lon-mediated pdt degradation. Inducible transcription provides control of mf-Lon-mediated degradation of GFP-pdt#3 across a range of ATc induction levels. Fusion of the E. coli ssrA tag variants ec-AAV and ec-ASV to mf-Lon shift the GFP degradation profile, and inactivation of mf-Lon protease activity (S692A) blocks GFP degradation. Data were collected 6 hours after ATc induction using GFP-pdt#3 as the degradation target. For all panels, the data show the mean of at least three biological replicates and the error bars show the standard deviation.
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Figure 2: Pdt system characterization(a) Comparative analysis of pdt-mediated degradation of mCherry and GFP. Pdt letter variants were fused to GFP and mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (50 ng/ml ATc for 6 h). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=1.09× – 0.01 with an R2 value of 0.98 and standard errors of 0.03 and 0.01 for the slope and y-intercept, respectively. (b) Pdt-dependent degradation of mCherry in L. lactis. Nisin induced mf-Lon expression in L. lactis causes pdt-dependent mCherry degradation. Data show the geometric mean fluorescence as a percent of the fluorescence of uninduced cells. Nisin induction was 3 ng/ml. (c) Comparative analysis of pdt letter variants in E. coli and L. lactis. Pdt letter variants were fused to mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (6 hour induction, E. coli: 50 ng/ml ATc and L. lactis: 3 ng/ml nisin). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=0.51× – 0.04 with an R2 value of 0.91 and standard errors of 0.03 and 0.02 for the slope and y-intercept, respectively. (d) Transcription and post-translation-based control of mf-Lon-mediated pdt degradation. Inducible transcription provides control of mf-Lon-mediated degradation of GFP-pdt#3 across a range of ATc induction levels. Fusion of the E. coli ssrA tag variants ec-AAV and ec-ASV to mf-Lon shift the GFP degradation profile, and inactivation of mf-Lon protease activity (S692A) blocks GFP degradation. Data were collected 6 hours after ATc induction using GFP-pdt#3 as the degradation target. For all panels, the data show the mean of at least three biological replicates and the error bars show the standard deviation.

Mentions: To determine if this GFP-pdt characterization can be used to predict pdt-mediated degradation of other protein targets, we placed pdt variants on the fluorescent protein mCherry and measured degradation following mf-Lon induction. As seen in Figure 2a, the letter variants produced mCherry degradation dynamics that correlated strongly with GFP degradation, displaying linear regression with an R2 value of 0.98. The slope of the regression line (1.09) and its y-intercept (−0.01) suggest that mf-Lon-mediated degradation of GFP and mCherry occurred at similar relative rates for all pdt letter variants tested. Pdt number variants also showed strong correlation for mCherry and GFP, with a linear regression R2 value of 0.97 (Supplementary Fig. 4a).


Tunable protein degradation in bacteria.

Cameron DE, Collins JJ - Nat. Biotechnol. (2014)

Pdt system characterization(a) Comparative analysis of pdt-mediated degradation of mCherry and GFP. Pdt letter variants were fused to GFP and mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (50 ng/ml ATc for 6 h). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=1.09× – 0.01 with an R2 value of 0.98 and standard errors of 0.03 and 0.01 for the slope and y-intercept, respectively. (b) Pdt-dependent degradation of mCherry in L. lactis. Nisin induced mf-Lon expression in L. lactis causes pdt-dependent mCherry degradation. Data show the geometric mean fluorescence as a percent of the fluorescence of uninduced cells. Nisin induction was 3 ng/ml. (c) Comparative analysis of pdt letter variants in E. coli and L. lactis. Pdt letter variants were fused to mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (6 hour induction, E. coli: 50 ng/ml ATc and L. lactis: 3 ng/ml nisin). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=0.51× – 0.04 with an R2 value of 0.91 and standard errors of 0.03 and 0.02 for the slope and y-intercept, respectively. (d) Transcription and post-translation-based control of mf-Lon-mediated pdt degradation. Inducible transcription provides control of mf-Lon-mediated degradation of GFP-pdt#3 across a range of ATc induction levels. Fusion of the E. coli ssrA tag variants ec-AAV and ec-ASV to mf-Lon shift the GFP degradation profile, and inactivation of mf-Lon protease activity (S692A) blocks GFP degradation. Data were collected 6 hours after ATc induction using GFP-pdt#3 as the degradation target. For all panels, the data show the mean of at least three biological replicates and the error bars show the standard deviation.
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Figure 2: Pdt system characterization(a) Comparative analysis of pdt-mediated degradation of mCherry and GFP. Pdt letter variants were fused to GFP and mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (50 ng/ml ATc for 6 h). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=1.09× – 0.01 with an R2 value of 0.98 and standard errors of 0.03 and 0.01 for the slope and y-intercept, respectively. (b) Pdt-dependent degradation of mCherry in L. lactis. Nisin induced mf-Lon expression in L. lactis causes pdt-dependent mCherry degradation. Data show the geometric mean fluorescence as a percent of the fluorescence of uninduced cells. Nisin induction was 3 ng/ml. (c) Comparative analysis of pdt letter variants in E. coli and L. lactis. Pdt letter variants were fused to mCherry, and the percent fluorescence remaining after mf-Lon induction is shown (6 hour induction, E. coli: 50 ng/ml ATc and L. lactis: 3 ng/ml nisin). Fluorescent data were collected by flow cytometry, and the pdt variants shown are pdt#3, #3A, #3B, #3C, #3D, #3E, listed in order of increasing percent fluorescence. The best-fit line by linear regression is y=0.51× – 0.04 with an R2 value of 0.91 and standard errors of 0.03 and 0.02 for the slope and y-intercept, respectively. (d) Transcription and post-translation-based control of mf-Lon-mediated pdt degradation. Inducible transcription provides control of mf-Lon-mediated degradation of GFP-pdt#3 across a range of ATc induction levels. Fusion of the E. coli ssrA tag variants ec-AAV and ec-ASV to mf-Lon shift the GFP degradation profile, and inactivation of mf-Lon protease activity (S692A) blocks GFP degradation. Data were collected 6 hours after ATc induction using GFP-pdt#3 as the degradation target. For all panels, the data show the mean of at least three biological replicates and the error bars show the standard deviation.
Mentions: To determine if this GFP-pdt characterization can be used to predict pdt-mediated degradation of other protein targets, we placed pdt variants on the fluorescent protein mCherry and measured degradation following mf-Lon induction. As seen in Figure 2a, the letter variants produced mCherry degradation dynamics that correlated strongly with GFP degradation, displaying linear regression with an R2 value of 0.98. The slope of the regression line (1.09) and its y-intercept (−0.01) suggest that mf-Lon-mediated degradation of GFP and mCherry occurred at similar relative rates for all pdt letter variants tested. Pdt number variants also showed strong correlation for mCherry and GFP, with a linear regression R2 value of 0.97 (Supplementary Fig. 4a).

Bottom Line: Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli.We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria.We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Boston University, Boston, Massachusetts, USA. [2] Center of Synthetic Biology, Boston University, Boston, Massachusetts, USA. [3] Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

ABSTRACT
Tunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.

Show MeSH
Related in: MedlinePlus