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Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.

ABSTRACT: BACKGROUND: Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products. RESULTS: Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed beta-lactamase (DeltaW290) selection cassette contains a segment inside the beta-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests. CONCLUSIONS: Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway or ligase-independent cloning (LIC) .

SUBMITTER: Olieric N

PROVIDER: S-EPMC2924254 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.

Olieric Natacha N Kuchen Melanie M Wagen Sandro S Sauter Marion M Crone Stephanie S Edmondson Sonia S Frey Daniel D Ostermeier Christian C Steinmetz Michel O MO Jaussi Rolf R

BMC biotechnology 20100809

<h4>Background</h4>Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.<h4>Results</h4>Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expr ...[more]

PMID: 20691119

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Project description:BackgroundTagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar).ResultsHere we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in frame into all 40 plasmids using blunt end or TA ligations, requiring only verification of the orientation. We have established a RSFC vector family for the commonly used protein expression host Pichia pastoris and demonstrated the system with the secretory expression of horseradish peroxidase (HRP). HRP fusions to four tags (Myc, FLAG, His, Strep) and two fusion proteins (GFP and MBP) showed a 31-fold difference in volumetric activities. C-terminal tagging caused in some cases almost a complete loss of function, whereas N-terminal tags showed moderate differences.ConclusionsThe RSFC vectors provide an unprecedented toolbox for expression optimization in P. pastoris. The results obtained with HRP underline the importance of comparing different tags to maximize activities of fusion proteins. In a similar fashion the RSFC strategy can be applied in other expression hosts to screen for optimal promoters, signal sequences or to facilitate the evaluation of (iso-) enzyme families.

Project description:BACKGROUND:When creating plant transformation vectors, full control of nucleotides flanking the insert in the final construct may be desirable. Modern ligase-independent methods for DNA-recombination are based on linearization by classical type II restriction endonucleases (REs) alone or in combination with nicking enzymes leaving residual nucleotides behind in the final construct. We here explore the use of type IIS and type IIB REs for vector linearization that combined with sequence and ligase-independent cloning (SLIC) overcomes this problem and promotes seamless gene-insertion in vectors. Providing the basis for a collection of biolistic plant transformation vectors ready to be cloned with different genes-of-interest, we present two vectors, where promoter and terminator are joined by a spacer. During spacer-removal linearization (SRL), type IIS and type IIB REs remove their own recognition sequences from the vector leaving no undesired, short sequences behind. RESULTS:We designed two plant transformation vectors prepared for SRL in combination with SLIC, pAUrumII and pAUrumIII, harboring a spacer with recognition sites for a type IIS and IIB RE, respectively. The gene for a green fluorescent protein, gfp, was successfully cloned into both vectors; traces of pAUrumIII, however, contaminated the transformation due to incomplete linearization, an issue not encountered with the type IIS linearized pAUrumII. Both constructs, pAUrumII-gfp and pAUrumIII-gfp, were functional, when tested in vitro on wheat and barley endosperm cells for transient gfp expression. CONCLUSIONS:All nucleotides flanking an insert in a biolistic plant transformation vector can be customized by means of SRL in combination with SLIC. Especially type IIS REs promote an efficient cloning result. Based on our findings, we believe that the SRL system can be useful in a series of plant transformation vectors, favoring the presence of functional sequences for optimal expression over redundant cloning-site remnants.

Dataset Information

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.

Publications

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.

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