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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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(A) A small set of RBS sequences was designed to span several orders of magnitude of protein expression. The RBS set was composed of six pre-characterized RBS core sequences flanked by constant upstream and downstream insulators that were paired to the genes of interest, as detailed in Supplementary Figure S4 and Supplementary Methods. (B) Flow cytometry fluorescence measurements of cells expressing YFP, where in each clone a different RBS sequence (A–F) was located upstream to the coding sequence. (−) represents the autofluorescence of cells when no fluorescent protein is expressed.
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gkt151-F2: (A) A small set of RBS sequences was designed to span several orders of magnitude of protein expression. The RBS set was composed of six pre-characterized RBS core sequences flanked by constant upstream and downstream insulators that were paired to the genes of interest, as detailed in Supplementary Figure S4 and Supplementary Methods. (B) Flow cytometry fluorescence measurements of cells expressing YFP, where in each clone a different RBS sequence (A–F) was located upstream to the coding sequence. (−) represents the autofluorescence of cells when no fluorescent protein is expressed.

Mentions: As RBS expression modulators we selected six sequences that were previously demonstrated by Salis et al. to span several orders of magnitude in protein expression levels (9) (Figure 2A). These synthetic DNA sequences are composed of three distinct functional parts: (i) a spacer sequence (5′-UTR), (ii) RBS core, which can also be referred to as a modified Shine–Dalgarno sequence, and (iii) the ATG starting codon followed by 6His-tag encoded in the N-terminus of the protein. Distinct RBS expression modulators contain a modified Shine–Dalgarno sequence as an RBS core, but they are flanked by identical upstream and downstream insulator sequences.Figure 2.


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)

(A) A small set of RBS sequences was designed to span several orders of magnitude of protein expression. The RBS set was composed of six pre-characterized RBS core sequences flanked by constant upstream and downstream insulators that were paired to the genes of interest, as detailed in Supplementary Figure S4 and Supplementary Methods. (B) Flow cytometry fluorescence measurements of cells expressing YFP, where in each clone a different RBS sequence (A–F) was located upstream to the coding sequence. (−) represents the autofluorescence of cells when no fluorescent protein is expressed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt151-F2: (A) A small set of RBS sequences was designed to span several orders of magnitude of protein expression. The RBS set was composed of six pre-characterized RBS core sequences flanked by constant upstream and downstream insulators that were paired to the genes of interest, as detailed in Supplementary Figure S4 and Supplementary Methods. (B) Flow cytometry fluorescence measurements of cells expressing YFP, where in each clone a different RBS sequence (A–F) was located upstream to the coding sequence. (−) represents the autofluorescence of cells when no fluorescent protein is expressed.
Mentions: As RBS expression modulators we selected six sequences that were previously demonstrated by Salis et al. to span several orders of magnitude in protein expression levels (9) (Figure 2A). These synthetic DNA sequences are composed of three distinct functional parts: (i) a spacer sequence (5′-UTR), (ii) RBS core, which can also be referred to as a modified Shine–Dalgarno sequence, and (iii) the ATG starting codon followed by 6His-tag encoded in the N-terminus of the protein. Distinct RBS expression modulators contain a modified Shine–Dalgarno sequence as an RBS core, but they are flanked by identical upstream and downstream insulator sequences.Figure 2.

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