The Experts below are selected from a list of 12102 Experts worldwide ranked by ideXlab platform
Jaeyeon Jeong - One of the best experts on this subject based on the ideXlab platform.
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optimizing t4 dna polymerase conditions enhances the efficiency of one step sequence and ligation independent Cloning
BioTechniques, 2017Co-Authors: Mohammad Nazrul Islam, Kyeong Won Lee, Hyungsoon Yim, Seong Hyuk Lee, Haechang Jung, Junghyun Lee, Jaeyeon JeongAbstract:Previously, we developed a one-step sequence- and Ligation-Independent Cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its clon...
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one step sequence and ligation independent Cloning as a rapid and versatile Cloning method for functional genomics studies
Applied and Environmental Microbiology, 2012Co-Authors: Jaeyeon Jeong, Hyungsoon Yim, Junghyun Lee, Jiyoung Ryu, Hyun Sook Lee, Dongseung Seen, Sung Gyun KangAbstract:We developed one-step sequence- and Ligation-Independent Cloning (SLIC) as a simple, cost-effective, time-saving, and versatile Cloning method. Highly efficient and directional Cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.
Stephen J Elledge - One of the best experts on this subject based on the ideXlab platform.
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slic a method for sequence and ligation independent Cloning
Methods of Molecular Biology, 2012Co-Authors: Stephen J ElledgeAbstract:We describe here a method for sequence- and Ligation-Independent Cloning (SLIC). SLIC uses an exonuclease, T4 DNA polymerase, to generate single-stranded DNA overhangs in insert and vector sequences. These fragments are then assembled in vitro and transformed into Escherichia coli to generate recombinant DNA of interest. SLIC inserts can also be generated by incomplete PCR (iPCR) or mixed PCR. As many as five inserts can be assembled in one reaction simultaneously with great efficiency using SLIC. SLIC circumvents sequence constraints for recombinant DNA using standard restriction enzyme-mediated Cloning and previous Ligation-Independent Cloning methods and provides a new approach for the efficient generation of recombinant DNA.
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harnessing homologous recombination in vitro to generate recombinant dna via slic
Nature Methods, 2007Co-Authors: Mamie Z Li, Stephen J ElledgeAbstract:We describe a new Cloning method, sequence and ligation–independent Cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
Lucy Stols - One of the best experts on this subject based on the ideXlab platform.
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New vectors for co-expression of proteins: Structure of Bacillus subtilis ScoAB obtained by high-throughput protocols☆
Protein Expression and Purification, 2007Co-Authors: Lucy Stols, William H. Eschenfeldt, Cynthia Sanville Millard, Frank R. Collart, Min Zhou, J. Abdullah, Mark I. DonnellyAbstract:Abstract The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, Ligation-Independent Cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, Ligation-Independent Cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8–15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.
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a new vector for high throughput ligation independent Cloning encoding a tobacco etch virus protease cleavage site
Protein Expression and Purification, 2002Co-Authors: Lucy Stols, Frank R. Collart, Minyi Gu, Lynda Dieckman, Rosemarie Raffen, Mark I. DonnellyAbstract:To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for Cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-Independent Cloning (LIC) is well suited to robotic Cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high Cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.
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a new vector for high throughput ligation independent Cloning encoding a tobacco etch virus protease cleavage site
Protein Expression and Purification, 2002Co-Authors: Lucy Stols, Frank R. Collart, Lynda Dieckman, Rosemarie Raffen, Mark DonnellyAbstract:To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for Cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-Independent Cloning (LIC) is well suited to robotic Cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high Cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.
Mark I. Donnelly - One of the best experts on this subject based on the ideXlab platform.
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New vectors for co-expression of proteins: Structure of Bacillus subtilis ScoAB obtained by high-throughput protocols☆
Protein Expression and Purification, 2007Co-Authors: Lucy Stols, William H. Eschenfeldt, Cynthia Sanville Millard, Frank R. Collart, Min Zhou, J. Abdullah, Mark I. DonnellyAbstract:Abstract The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, Ligation-Independent Cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, Ligation-Independent Cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8–15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.
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a new vector for high throughput ligation independent Cloning encoding a tobacco etch virus protease cleavage site
Protein Expression and Purification, 2002Co-Authors: Lucy Stols, Frank R. Collart, Minyi Gu, Lynda Dieckman, Rosemarie Raffen, Mark I. DonnellyAbstract:To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for Cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-Independent Cloning (LIC) is well suited to robotic Cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high Cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.
Rosario Munoz - One of the best experts on this subject based on the ideXlab platform.
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the puri family of expression vectors a versatile set of ligation independent Cloning plasmids for producing recombinant his fusion proteins
Protein Expression and Purification, 2011Co-Authors: Jose Antonio Curiel, Blanca De Las Rivas, Jose M Mancheno, Rosario MunozAbstract:A family of restriction enzyme- and Ligation-Independent Cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the milligram scale. The newly designed vectors combines two different promoters (lpp(p)-5 and T7 RNA polymerase O10), two different endoprotease recognition sites for the His₆-tag removal (enterokinase and tobacco etch virus), different antibiotic selectable markers (ampicillin and erythromycin resistance), and different placements of the His₆-tag (N- and C-terminus). A single gene can be cloned and further expressed in the eight pURI vectors by using six nucleotide primers, avoiding the restriction enzyme and ligation steps. A unique NotI site was introduced to facilitate the selection of the recombinant plasmid. As a case study, the new vectors have been used to clone the gene coding for the phenolic acid decarboxylase from Lactobacillus plantarum. Interestingly, the obtained results revealed markedly different production levels of the target protein, emphasizing the relevance of the Cloning strategy on soluble protein production yield. Efficient purification and tag removal steps showed that the affinity tag and the protease cleavage sites functioned properly. The novel family of pURI vectors designed for parallel Cloning is a useful and versatile tool for the production and purification of a protein of interest.
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expression vectors for enzyme restriction and ligation independent Cloning for producing recombinant his fusion proteins
Biotechnology Progress, 2008Co-Authors: Blanca De Las Rivas, Jose Antonio Curiel, Jose M Mancheno, Rosario MunozAbstract:In this work we have constructed two novel expression vectors, designated as pURI2 and pURI3, which enable parallel Cloning of a given target gene for producing recombinant His-fusion proteins. The vectors were created using the well-known pT7-7 and pIN-III-A3 plasmids as their template. The same DNA fragment containing the His-tag, enterokinase cleavage site, and a NotI unique site, as well as keeping the HindIII unique restriction site, was introduced in both vectors. These vectors have been designed to avoid the enzyme restriction and ligation steps during the Cloning. The unique NotI site was introduced to facilitate the selection of the adequate recombinant plasmid. Parallel Cloning of the same polymerase chain reaction fragment can be carried out since both vectors shared the same leader sequence. The described strategy avoids tedious Cloning efforts into different expression vectors and represents a highly efficient means of Cloning. To validate our vectors, we have cloned one target gene in both vectors and used expression and purification techniques to obtain the recombinant target protein. We herein show that both vectors function effectively in all the required experimental steps-Cloning, expression, purification, and cleavage.