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Standards not that standard.

Vilanova C, Tanner K, Dorado-Morales P, Villaescusa P, Chugani D, Frías A, Segredo E, Molero X, Fritschi M, Morales L, Ramón D, Peña C, Peretó J, Porcar M - J Biol Eng (2015)

Bottom Line: In two consecutive letters to this journal, suggestions on the assembly methods for the Registry of standard biological parts have been described.We fully agree with those authors on the need of a more flexible building strategy and we highlight in the present work two major functional challenges standardization efforts have to deal with: the need of both universal and orthogonal behaviors.We provide experimental data that clearly indicate that such engineering requirements should not be taken for granted in Synthetic Biology.

View Article: PubMed Central - PubMed

Affiliation: Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, C. Catedràtic José Beltrán 2, 46980 Paterna, Spain.

ABSTRACT
There is a general assent on the key role of standards in Synthetic Biology. In two consecutive letters to this journal, suggestions on the assembly methods for the Registry of standard biological parts have been described. We fully agree with those authors on the need of a more flexible building strategy and we highlight in the present work two major functional challenges standardization efforts have to deal with: the need of both universal and orthogonal behaviors. We provide experimental data that clearly indicate that such engineering requirements should not be taken for granted in Synthetic Biology.

No MeSH data available.


Related in: MedlinePlus

Orthogonality tests performed on a simple combination of two Biobrick parts. a Fluorescence output displayed by E. coli XL1 strain transformed with a single plasmid containing a constitutive promoter coupled to a green fluorescent protein (Bb1), a single plasmid containing the same promoter coupled to a red fluorescent protein (Bb2), and a combination of both plasmids. Plots showing flow cytometry measurements performed on individual cells (dots). b Comparison of the proteomic profile of an E. coli strain constitutively expressing a green fluorescent protein (green lines) with that of the same strain carrying an empty plasmid (red lines) and the control non-transformed strain (blue lines). Proteins showing a statistically significant change in expression are numbered according to Additional file 1: Table S2
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Fig2: Orthogonality tests performed on a simple combination of two Biobrick parts. a Fluorescence output displayed by E. coli XL1 strain transformed with a single plasmid containing a constitutive promoter coupled to a green fluorescent protein (Bb1), a single plasmid containing the same promoter coupled to a red fluorescent protein (Bb2), and a combination of both plasmids. Plots showing flow cytometry measurements performed on individual cells (dots). b Comparison of the proteomic profile of an E. coli strain constitutively expressing a green fluorescent protein (green lines) with that of the same strain carrying an empty plasmid (red lines) and the control non-transformed strain (blue lines). Proteins showing a statistically significant change in expression are numbered according to Additional file 1: Table S2

Mentions: We bench-tested these engineering pillars in the simplest scenarios: standardization was studied by introducing six DNA constructions (see Additional file 1: Table S1) built from commonly-used Biobrick parts in six different laboratory strains of E. coli and measuring their output under the same experimental conditions, whereas orthogonality was tested by co-transforming one of the strains (XL1-Blue) with a couple of these constructions (a green fluorescent protein placed under the control of a constitutive promoter, and a red fluorescent protein controlled by the same promoter) and measuring their output with flow cytometry techniques. Under our experimental conditions, significant differences in terms of expression levels were found among all the strains in five out of six constructions (Fig. 1) regardless the promoter type (constitutive or inducible) and the reporter protein (fluorescent proteins or β-galactosidase), and double transformants did not exhibit a 1:1 red:green fluorescent phenotype (Fig. 2a). The lack of orthogonality of these two biological parts between them was in contrast with the stability of E. coli as a chassis, as we tested through a proteomics approach. Figure 2b shows the proteomic profile of a transformed E. coli strain with a GFP-containing plasmid and of two control strains (one non-transformed and one containing the empty plasmid), which reveals a minor impact of GFP and/or antibiotic resistance expression on the global bacterial proteomic architecture. E. coli is thus –at least in our conditions– a solid, orthogonal system respect to the heterologous protein expression shuttle it hosts.Fig. 1


Standards not that standard.

Vilanova C, Tanner K, Dorado-Morales P, Villaescusa P, Chugani D, Frías A, Segredo E, Molero X, Fritschi M, Morales L, Ramón D, Peña C, Peretó J, Porcar M - J Biol Eng (2015)

Orthogonality tests performed on a simple combination of two Biobrick parts. a Fluorescence output displayed by E. coli XL1 strain transformed with a single plasmid containing a constitutive promoter coupled to a green fluorescent protein (Bb1), a single plasmid containing the same promoter coupled to a red fluorescent protein (Bb2), and a combination of both plasmids. Plots showing flow cytometry measurements performed on individual cells (dots). b Comparison of the proteomic profile of an E. coli strain constitutively expressing a green fluorescent protein (green lines) with that of the same strain carrying an empty plasmid (red lines) and the control non-transformed strain (blue lines). Proteins showing a statistically significant change in expression are numbered according to Additional file 1: Table S2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4591577&req=5

Fig2: Orthogonality tests performed on a simple combination of two Biobrick parts. a Fluorescence output displayed by E. coli XL1 strain transformed with a single plasmid containing a constitutive promoter coupled to a green fluorescent protein (Bb1), a single plasmid containing the same promoter coupled to a red fluorescent protein (Bb2), and a combination of both plasmids. Plots showing flow cytometry measurements performed on individual cells (dots). b Comparison of the proteomic profile of an E. coli strain constitutively expressing a green fluorescent protein (green lines) with that of the same strain carrying an empty plasmid (red lines) and the control non-transformed strain (blue lines). Proteins showing a statistically significant change in expression are numbered according to Additional file 1: Table S2
Mentions: We bench-tested these engineering pillars in the simplest scenarios: standardization was studied by introducing six DNA constructions (see Additional file 1: Table S1) built from commonly-used Biobrick parts in six different laboratory strains of E. coli and measuring their output under the same experimental conditions, whereas orthogonality was tested by co-transforming one of the strains (XL1-Blue) with a couple of these constructions (a green fluorescent protein placed under the control of a constitutive promoter, and a red fluorescent protein controlled by the same promoter) and measuring their output with flow cytometry techniques. Under our experimental conditions, significant differences in terms of expression levels were found among all the strains in five out of six constructions (Fig. 1) regardless the promoter type (constitutive or inducible) and the reporter protein (fluorescent proteins or β-galactosidase), and double transformants did not exhibit a 1:1 red:green fluorescent phenotype (Fig. 2a). The lack of orthogonality of these two biological parts between them was in contrast with the stability of E. coli as a chassis, as we tested through a proteomics approach. Figure 2b shows the proteomic profile of a transformed E. coli strain with a GFP-containing plasmid and of two control strains (one non-transformed and one containing the empty plasmid), which reveals a minor impact of GFP and/or antibiotic resistance expression on the global bacterial proteomic architecture. E. coli is thus –at least in our conditions– a solid, orthogonal system respect to the heterologous protein expression shuttle it hosts.Fig. 1

Bottom Line: In two consecutive letters to this journal, suggestions on the assembly methods for the Registry of standard biological parts have been described.We fully agree with those authors on the need of a more flexible building strategy and we highlight in the present work two major functional challenges standardization efforts have to deal with: the need of both universal and orthogonal behaviors.We provide experimental data that clearly indicate that such engineering requirements should not be taken for granted in Synthetic Biology.

View Article: PubMed Central - PubMed

Affiliation: Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, C. Catedràtic José Beltrán 2, 46980 Paterna, Spain.

ABSTRACT
There is a general assent on the key role of standards in Synthetic Biology. In two consecutive letters to this journal, suggestions on the assembly methods for the Registry of standard biological parts have been described. We fully agree with those authors on the need of a more flexible building strategy and we highlight in the present work two major functional challenges standardization efforts have to deal with: the need of both universal and orthogonal behaviors. We provide experimental data that clearly indicate that such engineering requirements should not be taken for granted in Synthetic Biology.

No MeSH data available.


Related in: MedlinePlus