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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 modular cloning strategy for combinatorial assembly of multi-gene constructs. During the iterative assembly process, each gene of interest is joined with a chloramphenicol (Cm) resistance cassette and paired with the library of RBS sequences. To incorporate the next target gene, the marker is discarded, and the additional part is assembled into the vector. The newly formed construct contains the two RBS-modified genes and a resistance marker, enabling once again direct selection for positive constructs. This sequence of steps can be repeated to easily assemble a combinatorial library of RBS-modulated multi-gene operons.
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gkt151-F3: A modular cloning strategy for combinatorial assembly of multi-gene constructs. During the iterative assembly process, each gene of interest is joined with a chloramphenicol (Cm) resistance cassette and paired with the library of RBS sequences. To incorporate the next target gene, the marker is discarded, and the additional part is assembled into the vector. The newly formed construct contains the two RBS-modified genes and a resistance marker, enabling once again direct selection for positive constructs. This sequence of steps can be repeated to easily assemble a combinatorial library of RBS-modulated multi-gene operons.

Mentions: To facilitate the library construction process, we used an augmented BioBrick (14) cloning strategy designated for the assembly of synthetic operons (Figure 3). In this procedure, genetic parts are iteratively assembled using a positive-selection stage that bypasses the need for time-consuming screening steps. Briefly, a chloramphenicol (Cm) resistance cassette is joined to all of the genetic parts that are to be assembled. In each step, an additional genetic part is incorporated into the construct while the resistance cassette enables a direct selection for properly assembled constructs. The vector is then ‘recycled’ for the next iteration by excising the resistance cassette. The resulting library of operons is then transformed into cells and screened for a desired phenotype. Inference of the RBS composition across the operon in a specific clone is performed by sequencing a barcode located at the 3′-UTR of the operon. The barcode is generated during the assembly process by iteratively concatenating a short identifying sequence onto the 3′-UTR of the operon (Supplementary Methods and Supplementary Figures S9 and S10). Each genetic variant in the library contains a distinct barcode sequence from which the RBS composition of all the genes in the operon can be inferred in a single sequencing reaction (Supplementary Tables S1 and S2).Figure 3.


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 modular cloning strategy for combinatorial assembly of multi-gene constructs. During the iterative assembly process, each gene of interest is joined with a chloramphenicol (Cm) resistance cassette and paired with the library of RBS sequences. To incorporate the next target gene, the marker is discarded, and the additional part is assembled into the vector. The newly formed construct contains the two RBS-modified genes and a resistance marker, enabling once again direct selection for positive constructs. This sequence of steps can be repeated to easily assemble a combinatorial library of RBS-modulated multi-gene operons.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt151-F3: A modular cloning strategy for combinatorial assembly of multi-gene constructs. During the iterative assembly process, each gene of interest is joined with a chloramphenicol (Cm) resistance cassette and paired with the library of RBS sequences. To incorporate the next target gene, the marker is discarded, and the additional part is assembled into the vector. The newly formed construct contains the two RBS-modified genes and a resistance marker, enabling once again direct selection for positive constructs. This sequence of steps can be repeated to easily assemble a combinatorial library of RBS-modulated multi-gene operons.
Mentions: To facilitate the library construction process, we used an augmented BioBrick (14) cloning strategy designated for the assembly of synthetic operons (Figure 3). In this procedure, genetic parts are iteratively assembled using a positive-selection stage that bypasses the need for time-consuming screening steps. Briefly, a chloramphenicol (Cm) resistance cassette is joined to all of the genetic parts that are to be assembled. In each step, an additional genetic part is incorporated into the construct while the resistance cassette enables a direct selection for properly assembled constructs. The vector is then ‘recycled’ for the next iteration by excising the resistance cassette. The resulting library of operons is then transformed into cells and screened for a desired phenotype. Inference of the RBS composition across the operon in a specific clone is performed by sequencing a barcode located at the 3′-UTR of the operon. The barcode is generated during the assembly process by iteratively concatenating a short identifying sequence onto the 3′-UTR of the operon (Supplementary Methods and Supplementary Figures S9 and S10). Each genetic variant in the library contains a distinct barcode sequence from which the RBS composition of all the genes in the operon can be inferred in a single sequencing reaction (Supplementary Tables S1 and S2).Figure 3.

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