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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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Carotenoid accumulation profile varies with the RBS sequences of biosynthetic genes. (A) We assembled a library of synthetic operons differing in the RBS sequences regulating each of the seven genes of the carotenoid biosynthesis pathway. (B) A binocular microscopy imaging of E. coli colonies transformed with the operon library. The color of the colony corresponds to the composition of the accumulated carotenoids, each having a characteristic color. Image was constructed by stitching multiple adjacent fields. (C) The carotenoid accumulation profile and RBS composition of clones isolated from the transformed library. The RBS composition of each clone was determined by sequencing (RBS encoding in barcode refers to the order of genes as illustrated in Figure 3A), and the carotenoid profile of each clone was analyzed using HPLC. Different genotypes result in distinct phenotypes, i.e. distinct carotenoid accumulation profiles. Circle area indicates the production yield of major carotenoid intermediates (>10% of total carotenoids), according to the metabolic pathway described on the right.
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gkt151-F5: Carotenoid accumulation profile varies with the RBS sequences of biosynthetic genes. (A) We assembled a library of synthetic operons differing in the RBS sequences regulating each of the seven genes of the carotenoid biosynthesis pathway. (B) A binocular microscopy imaging of E. coli colonies transformed with the operon library. The color of the colony corresponds to the composition of the accumulated carotenoids, each having a characteristic color. Image was constructed by stitching multiple adjacent fields. (C) The carotenoid accumulation profile and RBS composition of clones isolated from the transformed library. The RBS composition of each clone was determined by sequencing (RBS encoding in barcode refers to the order of genes as illustrated in Figure 3A), and the carotenoid profile of each clone was analyzed using HPLC. Different genotypes result in distinct phenotypes, i.e. distinct carotenoid accumulation profiles. Circle area indicates the production yield of major carotenoid intermediates (>10% of total carotenoids), according to the metabolic pathway described on the right.

Mentions: Fluorescence images of colonies expressing RBS-modulated variants of the tri-color reporter operon were taken using a Nikon ECLIPSE E800 microscope equipped with a Nikon Intensilight (C-HGFIE) for illumination. Chroma filter cubes were used to image fluorescence proteins: mCherry (excitation filter 530–560 nm, emission filter 590–650 nm, 30 ms exposure), cyan fluorescent protein (mCFP: excitation filter 426–446 nm, emission filter 460–500 nm, 60 ms exposure) and YFP (mYFP: excitation filter 490–510 nm, emission filter 520–550 nm, 800 ms exposure). Images were captured with a Nikon DS-5M-L1 digital Sight Camera System using the NIS-Elements BR3.22 software. Different channels were overlaid to give the figures shown. Images of colonies appearing in Figure 5B were taken using a binocular microscope (WILD M8; Heerbrugg, Switzerland) with Schott Ace Fiber Optic Light Source 150W Microscope Illuminator. Images were captured using a Nikon Digital Sight Camera System. Stitching of adjacent fields was done using AutoStitch software (http://www.cs.bath.ac.uk/brown/autostitch/autostitch.html).


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)

Carotenoid accumulation profile varies with the RBS sequences of biosynthetic genes. (A) We assembled a library of synthetic operons differing in the RBS sequences regulating each of the seven genes of the carotenoid biosynthesis pathway. (B) A binocular microscopy imaging of E. coli colonies transformed with the operon library. The color of the colony corresponds to the composition of the accumulated carotenoids, each having a characteristic color. Image was constructed by stitching multiple adjacent fields. (C) The carotenoid accumulation profile and RBS composition of clones isolated from the transformed library. The RBS composition of each clone was determined by sequencing (RBS encoding in barcode refers to the order of genes as illustrated in Figure 3A), and the carotenoid profile of each clone was analyzed using HPLC. Different genotypes result in distinct phenotypes, i.e. distinct carotenoid accumulation profiles. Circle area indicates the production yield of major carotenoid intermediates (>10% of total carotenoids), according to the metabolic pathway described on the right.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt151-F5: Carotenoid accumulation profile varies with the RBS sequences of biosynthetic genes. (A) We assembled a library of synthetic operons differing in the RBS sequences regulating each of the seven genes of the carotenoid biosynthesis pathway. (B) A binocular microscopy imaging of E. coli colonies transformed with the operon library. The color of the colony corresponds to the composition of the accumulated carotenoids, each having a characteristic color. Image was constructed by stitching multiple adjacent fields. (C) The carotenoid accumulation profile and RBS composition of clones isolated from the transformed library. The RBS composition of each clone was determined by sequencing (RBS encoding in barcode refers to the order of genes as illustrated in Figure 3A), and the carotenoid profile of each clone was analyzed using HPLC. Different genotypes result in distinct phenotypes, i.e. distinct carotenoid accumulation profiles. Circle area indicates the production yield of major carotenoid intermediates (>10% of total carotenoids), according to the metabolic pathway described on the right.
Mentions: Fluorescence images of colonies expressing RBS-modulated variants of the tri-color reporter operon were taken using a Nikon ECLIPSE E800 microscope equipped with a Nikon Intensilight (C-HGFIE) for illumination. Chroma filter cubes were used to image fluorescence proteins: mCherry (excitation filter 530–560 nm, emission filter 590–650 nm, 30 ms exposure), cyan fluorescent protein (mCFP: excitation filter 426–446 nm, emission filter 460–500 nm, 60 ms exposure) and YFP (mYFP: excitation filter 490–510 nm, emission filter 520–550 nm, 800 ms exposure). Images were captured with a Nikon DS-5M-L1 digital Sight Camera System using the NIS-Elements BR3.22 software. Different channels were overlaid to give the figures shown. Images of colonies appearing in Figure 5B were taken using a binocular microscope (WILD M8; Heerbrugg, Switzerland) with Schott Ace Fiber Optic Light Source 150W Microscope Illuminator. Images were captured using a Nikon Digital Sight Camera System. Stitching of adjacent fields was done using AutoStitch software (http://www.cs.bath.ac.uk/brown/autostitch/autostitch.html).

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