Limits...
Bottom-up engineering of biological systems through standard bricks: a modularity study on basic parts and devices.

Pasotti L, Politi N, Zucca S, Cusella De Angelis MG, Magni P - PLoS ONE (2012)

Bottom Line: If modularity persists, identical transcriptional signals trigger identical GFP outputs.Promoters activities (referred to a standard promoter) can vary when they are measured via different reporter devices (up to 22%), when they are used within a two-expression-cassette system (up to 35%) and when they drive another device in a functionally interconnected circuit (up to 44%).This paper provides a significant contribution to the study of modularity limitations in building biological systems by providing useful data on context-dependent variability of biological components.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy.

ABSTRACT

Background: Modularity is a crucial issue in the engineering world, as it enables engineers to achieve predictable outcomes when different components are interconnected. Synthetic Biology aims to apply key concepts of engineering to design and construct new biological systems that exhibit a predictable behaviour. Even if physical and measurement standards have been recently proposed to facilitate the assembly and characterization of biological components, real modularity is still a major research issue. The success of the bottom-up approach strictly depends on the clear definition of the limits in which biological functions can be predictable.

Results: The modularity of transcription-based biological components has been investigated in several conditions. First, the activity of a set of promoters was quantified in Escherichia coli via different measurement systems (i.e., different plasmids, reporter genes, ribosome binding sites) relative to an in vivo reference promoter. Second, promoter activity variation was measured when two independent gene expression cassettes were assembled in the same system. Third, the interchangeability of input modules (a set of constitutive promoters and two regulated promoters) connected to a fixed output device (a logic inverter) expressing GFP was evaluated. The three input modules provide tunable transcriptional signals that drive the output device. If modularity persists, identical transcriptional signals trigger identical GFP outputs. To verify this, all the input devices were individually characterized and then the input-output characteristic of the logic inverter was derived in the different configurations.

Conclusions: Promoters activities (referred to a standard promoter) can vary when they are measured via different reporter devices (up to 22%), when they are used within a two-expression-cassette system (up to 35%) and when they drive another device in a functionally interconnected circuit (up to 44%). This paper provides a significant contribution to the study of modularity limitations in building biological systems by providing useful data on context-dependent variability of biological components.

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Related in: MedlinePlus

Measured RPU values for the five investigated promoters.Promoters were individually characterized via three reporter devices: GFP32, RFP34 and RFP32. Error bars represent the standard deviation of the mean activity computed on three clones. For each promoter, statistical analysis was performed via ANOVA test to compare the RPU activities measured via the three different reporter devices. Promoters showing a statistical difference (P<0.05) in the mean activities among the three conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P0.05) are marked with a ‘-’ sign. Strains with  were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
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pone-0039407-g002: Measured RPU values for the five investigated promoters.Promoters were individually characterized via three reporter devices: GFP32, RFP34 and RFP32. Error bars represent the standard deviation of the mean activity computed on three clones. For each promoter, statistical analysis was performed via ANOVA test to compare the RPU activities measured via the three different reporter devices. Promoters showing a statistical difference (P<0.05) in the mean activities among the three conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P0.05) are marked with a ‘-’ sign. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).

Mentions: Results are shown in Figure 2 for the low copy condition. Promoters span a >10-fold RPU range, in which is the strongest one, while is the weakest one. Given a measurement system, the quantified activity of each promoter is reasonably reproducible among the technical replicates, giving an average coefficient of variation (CV) of 9%. As expected, promoters characterized via RFP34 give a higher absolute activity than with RFP32 (data not shown), as the BBa_B0032 RBS is weaker than BBa_B0034 [8], [15], [28]. Only activity is statistically different among the three tested measurement systems (P<0.05, ANOVA), yielding a CV of 22% among the three measured mean activities.


Bottom-up engineering of biological systems through standard bricks: a modularity study on basic parts and devices.

Pasotti L, Politi N, Zucca S, Cusella De Angelis MG, Magni P - PLoS ONE (2012)

Measured RPU values for the five investigated promoters.Promoters were individually characterized via three reporter devices: GFP32, RFP34 and RFP32. Error bars represent the standard deviation of the mean activity computed on three clones. For each promoter, statistical analysis was performed via ANOVA test to compare the RPU activities measured via the three different reporter devices. Promoters showing a statistical difference (P<0.05) in the mean activities among the three conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P0.05) are marked with a ‘-’ sign. Strains with  were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401228&req=5

pone-0039407-g002: Measured RPU values for the five investigated promoters.Promoters were individually characterized via three reporter devices: GFP32, RFP34 and RFP32. Error bars represent the standard deviation of the mean activity computed on three clones. For each promoter, statistical analysis was performed via ANOVA test to compare the RPU activities measured via the three different reporter devices. Promoters showing a statistical difference (P<0.05) in the mean activities among the three conditions are marked with a ‘+’ sign, while promoters not showing any significant difference (P0.05) are marked with a ‘-’ sign. Strains with were induced with 1 mM of isopropyl -D-1-thiogalactopyranoside (IPTG).
Mentions: Results are shown in Figure 2 for the low copy condition. Promoters span a >10-fold RPU range, in which is the strongest one, while is the weakest one. Given a measurement system, the quantified activity of each promoter is reasonably reproducible among the technical replicates, giving an average coefficient of variation (CV) of 9%. As expected, promoters characterized via RFP34 give a higher absolute activity than with RFP32 (data not shown), as the BBa_B0032 RBS is weaker than BBa_B0034 [8], [15], [28]. Only activity is statistically different among the three tested measurement systems (P<0.05, ANOVA), yielding a CV of 22% among the three measured mean activities.

Bottom Line: If modularity persists, identical transcriptional signals trigger identical GFP outputs.Promoters activities (referred to a standard promoter) can vary when they are measured via different reporter devices (up to 22%), when they are used within a two-expression-cassette system (up to 35%) and when they drive another device in a functionally interconnected circuit (up to 44%).This paper provides a significant contribution to the study of modularity limitations in building biological systems by providing useful data on context-dependent variability of biological components.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy.

ABSTRACT

Background: Modularity is a crucial issue in the engineering world, as it enables engineers to achieve predictable outcomes when different components are interconnected. Synthetic Biology aims to apply key concepts of engineering to design and construct new biological systems that exhibit a predictable behaviour. Even if physical and measurement standards have been recently proposed to facilitate the assembly and characterization of biological components, real modularity is still a major research issue. The success of the bottom-up approach strictly depends on the clear definition of the limits in which biological functions can be predictable.

Results: The modularity of transcription-based biological components has been investigated in several conditions. First, the activity of a set of promoters was quantified in Escherichia coli via different measurement systems (i.e., different plasmids, reporter genes, ribosome binding sites) relative to an in vivo reference promoter. Second, promoter activity variation was measured when two independent gene expression cassettes were assembled in the same system. Third, the interchangeability of input modules (a set of constitutive promoters and two regulated promoters) connected to a fixed output device (a logic inverter) expressing GFP was evaluated. The three input modules provide tunable transcriptional signals that drive the output device. If modularity persists, identical transcriptional signals trigger identical GFP outputs. To verify this, all the input devices were individually characterized and then the input-output characteristic of the logic inverter was derived in the different configurations.

Conclusions: Promoters activities (referred to a standard promoter) can vary when they are measured via different reporter devices (up to 22%), when they are used within a two-expression-cassette system (up to 35%) and when they drive another device in a functionally interconnected circuit (up to 44%). This paper provides a significant contribution to the study of modularity limitations in building biological systems by providing useful data on context-dependent variability of biological components.

Show MeSH
Related in: MedlinePlus