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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

Behaviour of a set of Biobrick parts in different E. coli host strains. The output of DNA constructs consisting of a promoter coupled to a reporter protein was measured under the same experimental conditions for each strain. Note that both constitutive and inducible promoters were tested, and different reporters (fluorescence proteins and coloured compounds) were used. All measurements were normalized by the OD600 value of each culture, and corrected by the basal output observed in control strains transformed with an empty plasmid. Error bars show the standard deviation of three independent biological replica. See Additional file 1 for further experimental details
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Fig1: Behaviour of a set of Biobrick parts in different E. coli host strains. The output of DNA constructs consisting of a promoter coupled to a reporter protein was measured under the same experimental conditions for each strain. Note that both constitutive and inducible promoters were tested, and different reporters (fluorescence proteins and coloured compounds) were used. All measurements were normalized by the OD600 value of each culture, and corrected by the basal output observed in control strains transformed with an empty plasmid. Error bars show the standard deviation of three independent biological replica. See Additional file 1 for further experimental details

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)

Behaviour of a set of Biobrick parts in different E. coli host strains. The output of DNA constructs consisting of a promoter coupled to a reporter protein was measured under the same experimental conditions for each strain. Note that both constitutive and inducible promoters were tested, and different reporters (fluorescence proteins and coloured compounds) were used. All measurements were normalized by the OD600 value of each culture, and corrected by the basal output observed in control strains transformed with an empty plasmid. Error bars show the standard deviation of three independent biological replica. See Additional file 1 for further experimental details
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Behaviour of a set of Biobrick parts in different E. coli host strains. The output of DNA constructs consisting of a promoter coupled to a reporter protein was measured under the same experimental conditions for each strain. Note that both constitutive and inducible promoters were tested, and different reporters (fluorescence proteins and coloured compounds) were used. All measurements were normalized by the OD600 value of each culture, and corrected by the basal output observed in control strains transformed with an empty plasmid. Error bars show the standard deviation of three independent biological replica. See Additional file 1 for further experimental details
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