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Spanning high-dimensional expression space using ribosome-binding site combinatorics.

Zelcbuch L, Antonovsky N, Bar-Even A, Levin-Karp A, Barenholz U, Dayagi M, Liebermeister W, Flamholz A, Noor E, Amram S, Brandis A, Bareia T, Yofe I, Jubran H, Milo R - Nucleic Acids Res. (2013)

Bottom Line: Protein levels are a dominant factor shaping natural and synthetic biological systems.By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude.We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

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RBS modulation of three fluorescent proteins spans a color space. (A) We combinatorially joined CFP, YFP and mCherry with three representatives of our RBS set (sequences ‘A’, ‘C’ and ‘E’) and assembled the genes together into a synthetic operon. The resulting operon library differs only in the RBS sequences regulating gene expression. (B) Fluorescence microscopy imaging of E. coli colonies, transformed with the operon library. The observed colors represent additive combinations of the three primary colors, assigned to each of the fluorescent proteins. Irregular colony shapes are the result of touching boundaries of adjacent colonies. Some colonies harboring weak RBS for all three fluorescent reporters appear black. Inset: a bright-field microscopy image. (C) Fluorescence imaging of E. coli colonies containing the tri-color RBS-modulated operon. The images are arranged on a 3D grid where the position on each axis corresponds to the RBS strength of the fluorescent protein. (D) YFP and mCherry accumulation rates of clones sampled from a two-color operon library. RBS composition, as determined by barcode sequencing, is shown. Identical genotypes (each labeled by a distinct color) cluster together in the fluorescence space. The effect of translational coupling is also evident, where higher protein accumulation rate of YFP modulates the accumulation rate of mCherry.
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gkt151-F4: RBS modulation of three fluorescent proteins spans a color space. (A) We combinatorially joined CFP, YFP and mCherry with three representatives of our RBS set (sequences ‘A’, ‘C’ and ‘E’) and assembled the genes together into a synthetic operon. The resulting operon library differs only in the RBS sequences regulating gene expression. (B) Fluorescence microscopy imaging of E. coli colonies, transformed with the operon library. The observed colors represent additive combinations of the three primary colors, assigned to each of the fluorescent proteins. Irregular colony shapes are the result of touching boundaries of adjacent colonies. Some colonies harboring weak RBS for all three fluorescent reporters appear black. Inset: a bright-field microscopy image. (C) Fluorescence imaging of E. coli colonies containing the tri-color RBS-modulated operon. The images are arranged on a 3D grid where the position on each axis corresponds to the RBS strength of the fluorescent protein. (D) YFP and mCherry accumulation rates of clones sampled from a two-color operon library. RBS composition, as determined by barcode sequencing, is shown. Identical genotypes (each labeled by a distinct color) cluster together in the fluorescence space. The effect of translational coupling is also evident, where higher protein accumulation rate of YFP modulates the accumulation rate of mCherry.

Mentions: To test whether RBS combinatorics can successfully span a multi-dimensional expression space, we constructed a tri-color reporter system. CFP, YFP and mCherry were randomly paired with three representatives of our RBS set (RBS sequences ‘A’, ‘C’ and ‘E’) and assembled together to yield a library of synthetic operons. The resulting library, therefore, contained 33 = 27 genetic variants, in which all three genes are in the same order but under the regulation of different RBS sequences (Figure 4A). On transformation, colonies display distinct color patterns (Figure 4B), resulting from differential expression of the fluorescent reporters.Figure 4.


Spanning high-dimensional expression space using ribosome-binding site combinatorics.

Zelcbuch L, Antonovsky N, Bar-Even A, Levin-Karp A, Barenholz U, Dayagi M, Liebermeister W, Flamholz A, Noor E, Amram S, Brandis A, Bareia T, Yofe I, Jubran H, Milo R - Nucleic Acids Res. (2013)

RBS modulation of three fluorescent proteins spans a color space. (A) We combinatorially joined CFP, YFP and mCherry with three representatives of our RBS set (sequences ‘A’, ‘C’ and ‘E’) and assembled the genes together into a synthetic operon. The resulting operon library differs only in the RBS sequences regulating gene expression. (B) Fluorescence microscopy imaging of E. coli colonies, transformed with the operon library. The observed colors represent additive combinations of the three primary colors, assigned to each of the fluorescent proteins. Irregular colony shapes are the result of touching boundaries of adjacent colonies. Some colonies harboring weak RBS for all three fluorescent reporters appear black. Inset: a bright-field microscopy image. (C) Fluorescence imaging of E. coli colonies containing the tri-color RBS-modulated operon. The images are arranged on a 3D grid where the position on each axis corresponds to the RBS strength of the fluorescent protein. (D) YFP and mCherry accumulation rates of clones sampled from a two-color operon library. RBS composition, as determined by barcode sequencing, is shown. Identical genotypes (each labeled by a distinct color) cluster together in the fluorescence space. The effect of translational coupling is also evident, where higher protein accumulation rate of YFP modulates the accumulation rate of mCherry.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643573&req=5

gkt151-F4: RBS modulation of three fluorescent proteins spans a color space. (A) We combinatorially joined CFP, YFP and mCherry with three representatives of our RBS set (sequences ‘A’, ‘C’ and ‘E’) and assembled the genes together into a synthetic operon. The resulting operon library differs only in the RBS sequences regulating gene expression. (B) Fluorescence microscopy imaging of E. coli colonies, transformed with the operon library. The observed colors represent additive combinations of the three primary colors, assigned to each of the fluorescent proteins. Irregular colony shapes are the result of touching boundaries of adjacent colonies. Some colonies harboring weak RBS for all three fluorescent reporters appear black. Inset: a bright-field microscopy image. (C) Fluorescence imaging of E. coli colonies containing the tri-color RBS-modulated operon. The images are arranged on a 3D grid where the position on each axis corresponds to the RBS strength of the fluorescent protein. (D) YFP and mCherry accumulation rates of clones sampled from a two-color operon library. RBS composition, as determined by barcode sequencing, is shown. Identical genotypes (each labeled by a distinct color) cluster together in the fluorescence space. The effect of translational coupling is also evident, where higher protein accumulation rate of YFP modulates the accumulation rate of mCherry.
Mentions: To test whether RBS combinatorics can successfully span a multi-dimensional expression space, we constructed a tri-color reporter system. CFP, YFP and mCherry were randomly paired with three representatives of our RBS set (RBS sequences ‘A’, ‘C’ and ‘E’) and assembled together to yield a library of synthetic operons. The resulting library, therefore, contained 33 = 27 genetic variants, in which all three genes are in the same order but under the regulation of different RBS sequences (Figure 4A). On transformation, colonies display distinct color patterns (Figure 4B), resulting from differential expression of the fluorescent reporters.Figure 4.

Bottom Line: Protein levels are a dominant factor shaping natural and synthetic biological systems.By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude.We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

Show MeSH
Related in: MedlinePlus