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SOMA: a single oligonucleotide mutagenesis and cloning approach.

Pfirrmann T, Lokapally A, Andréasson C, Ljungdahl P, Hollemann T - PLoS ONE (2013)

Bottom Line: We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos.In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms.As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Institute for Physiological Chemistry, Martin-Luther University Halle-Wittenberg, Halle, Germany. thorsten.pfirrmann@medizin.uni-halle.de

ABSTRACT
Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

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Generation of a semi-random mutagenized gene library using SOMA.(A) Ssy5 sequence with autolytic processing site (scissors) between A381 and A382 (blue). Replacement of conserved isoleucine378 (I; green) with aspartate (D; red) inactivates Ssy5. Primer D378X with random codon (NNN) theoretically capable of encoding all amino acids at position 378 (green). (B) Table with active D378 substitutions recovered in plasmids selected based on their ability to confer Ssy5-dependent growth on YPD+MM. (C) Dilutions of strain HKY77 (ssy5Δ) carrying pSH120 (SSY5; +), pRS316 (empty plasmid; -), I378G, I378D, I378S, I378F, I378V, or I378L.
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pone-0064870-g004: Generation of a semi-random mutagenized gene library using SOMA.(A) Ssy5 sequence with autolytic processing site (scissors) between A381 and A382 (blue). Replacement of conserved isoleucine378 (I; green) with aspartate (D; red) inactivates Ssy5. Primer D378X with random codon (NNN) theoretically capable of encoding all amino acids at position 378 (green). (B) Table with active D378 substitutions recovered in plasmids selected based on their ability to confer Ssy5-dependent growth on YPD+MM. (C) Dilutions of strain HKY77 (ssy5Δ) carrying pSH120 (SSY5; +), pRS316 (empty plasmid; -), I378G, I378D, I378S, I378F, I378V, or I378L.

Mentions: The activity of the S. cerevisiae Ssy5 protease is regulated in response to the availability of extracellular amino acids [27]. During its maturation, Ssy5 cleaves itself between alanine381 and alanine382 (Figure 4A, scissors). This is a requisite event for subsequent amino acid induced activation of the endoproteolytic activity of Ssy5 by proteolytic removal of the inhibitory N-terminal domain [28]. The transcription factors Stp1 and Stp2 are the only other known substrates of Ssy5, however, the cleavage site in these substrates is not known. We noted a highly conserved isoleucine (I; yellow) at position 378 of Ssy5 (Figure 4A). Based on the proximity of a conserved residue to the autolytic processing site (P4’ position), we applied SOMA to produce a gene library randomly mutagenized specifically at codon I378.


SOMA: a single oligonucleotide mutagenesis and cloning approach.

Pfirrmann T, Lokapally A, Andréasson C, Ljungdahl P, Hollemann T - PLoS ONE (2013)

Generation of a semi-random mutagenized gene library using SOMA.(A) Ssy5 sequence with autolytic processing site (scissors) between A381 and A382 (blue). Replacement of conserved isoleucine378 (I; green) with aspartate (D; red) inactivates Ssy5. Primer D378X with random codon (NNN) theoretically capable of encoding all amino acids at position 378 (green). (B) Table with active D378 substitutions recovered in plasmids selected based on their ability to confer Ssy5-dependent growth on YPD+MM. (C) Dilutions of strain HKY77 (ssy5Δ) carrying pSH120 (SSY5; +), pRS316 (empty plasmid; -), I378G, I378D, I378S, I378F, I378V, or I378L.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672168&req=5

pone-0064870-g004: Generation of a semi-random mutagenized gene library using SOMA.(A) Ssy5 sequence with autolytic processing site (scissors) between A381 and A382 (blue). Replacement of conserved isoleucine378 (I; green) with aspartate (D; red) inactivates Ssy5. Primer D378X with random codon (NNN) theoretically capable of encoding all amino acids at position 378 (green). (B) Table with active D378 substitutions recovered in plasmids selected based on their ability to confer Ssy5-dependent growth on YPD+MM. (C) Dilutions of strain HKY77 (ssy5Δ) carrying pSH120 (SSY5; +), pRS316 (empty plasmid; -), I378G, I378D, I378S, I378F, I378V, or I378L.
Mentions: The activity of the S. cerevisiae Ssy5 protease is regulated in response to the availability of extracellular amino acids [27]. During its maturation, Ssy5 cleaves itself between alanine381 and alanine382 (Figure 4A, scissors). This is a requisite event for subsequent amino acid induced activation of the endoproteolytic activity of Ssy5 by proteolytic removal of the inhibitory N-terminal domain [28]. The transcription factors Stp1 and Stp2 are the only other known substrates of Ssy5, however, the cleavage site in these substrates is not known. We noted a highly conserved isoleucine (I; yellow) at position 378 of Ssy5 (Figure 4A). Based on the proximity of a conserved residue to the autolytic processing site (P4’ position), we applied SOMA to produce a gene library randomly mutagenized specifically at codon I378.

Bottom Line: We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos.In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms.As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Institute for Physiological Chemistry, Martin-Luther University Halle-Wittenberg, Halle, Germany. thorsten.pfirrmann@medizin.uni-halle.de

ABSTRACT
Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

Show MeSH
Related in: MedlinePlus