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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Modularity study for input modules in interconnected systems.A) Schematic functional representation of the tested framework. B) Different input devices are assembled upstream of a tetR-based logic inverter. They provide transcriptional signals that drive the inverter. C) INPUT1: a set of four constitutive promoters of different strengths. D) INPUT2:  promoter, which is repressed by the endogenously-overexpressed lacI and can be induced by IPTG. E) INPUT3: luxR-based HSL-inducible device.  can be induced by LuxR-HSL complex. The luxR gene is produced by the weak basic activity of repressed  in absence of IPTG. IPTG = isopropyl -D-1-thiogalactopyranoside; HSL = N-3-oxohexanoyl-L-homoserine lactone.
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pone-0039407-g005: Modularity study for input modules in interconnected systems.A) Schematic functional representation of the tested framework. B) Different input devices are assembled upstream of a tetR-based logic inverter. They provide transcriptional signals that drive the inverter. C) INPUT1: a set of four constitutive promoters of different strengths. D) INPUT2: promoter, which is repressed by the endogenously-overexpressed lacI and can be induced by IPTG. E) INPUT3: luxR-based HSL-inducible device. can be induced by LuxR-HSL complex. The luxR gene is produced by the weak basic activity of repressed in absence of IPTG. IPTG = isopropyl -D-1-thiogalactopyranoside; HSL = N-3-oxohexanoyl-L-homoserine lactone.

Mentions: The modularity of biological devices was studied when dealing with functionally interconnected circuits. The basic idea driving this part of the study is illustrated in Figure 5A. Considering interconnected systems composed by different input blocks (X1, X2, …, XN) and a fixed output block (Z) downstream, if the signals provided by the input blocks are the same (in1 = in2 = … = inN), the output signals should be identical (out1 = out2 = … = outN) even if the input blocks are structurally different.


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)

Modularity study for input modules in interconnected systems.A) Schematic functional representation of the tested framework. B) Different input devices are assembled upstream of a tetR-based logic inverter. They provide transcriptional signals that drive the inverter. C) INPUT1: a set of four constitutive promoters of different strengths. D) INPUT2:  promoter, which is repressed by the endogenously-overexpressed lacI and can be induced by IPTG. E) INPUT3: luxR-based HSL-inducible device.  can be induced by LuxR-HSL complex. The luxR gene is produced by the weak basic activity of repressed  in absence of IPTG. IPTG = isopropyl -D-1-thiogalactopyranoside; HSL = N-3-oxohexanoyl-L-homoserine lactone.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039407-g005: Modularity study for input modules in interconnected systems.A) Schematic functional representation of the tested framework. B) Different input devices are assembled upstream of a tetR-based logic inverter. They provide transcriptional signals that drive the inverter. C) INPUT1: a set of four constitutive promoters of different strengths. D) INPUT2: promoter, which is repressed by the endogenously-overexpressed lacI and can be induced by IPTG. E) INPUT3: luxR-based HSL-inducible device. can be induced by LuxR-HSL complex. The luxR gene is produced by the weak basic activity of repressed in absence of IPTG. IPTG = isopropyl -D-1-thiogalactopyranoside; HSL = N-3-oxohexanoyl-L-homoserine lactone.
Mentions: The modularity of biological devices was studied when dealing with functionally interconnected circuits. The basic idea driving this part of the study is illustrated in Figure 5A. Considering interconnected systems composed by different input blocks (X1, X2, …, XN) and a fixed output block (Z) downstream, if the signals provided by the input blocks are the same (in1 = in2 = … = inN), the output signals should be identical (out1 = out2 = … = outN) even if the input blocks are structurally different.

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