Minimum Length of Homology Arms Required for Effective Red/ET Recombination

Fujimoto, Rika; Osakabe, Toshihiko; Saito, Masumichi; Kurosawa, Nobuyuki; Isobe, Masaharu

doi:10.1271/bbb.90584

Cited by 8 publications

(9 citation statements)

References 8 publications

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“…PCC 7002. Interestingly, this is more than 200 bp longer than the minimum homology arms reported for E. coli [44, 45], suggesting that homologous recombination may be less efficient in Synechococcus sp. PCC 7002 or that native exonuclease activity may degrade the linearized plasmid containing the integration cassette.…”

Section: Discussionmentioning

confidence: 86%

“…If the minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 can be reduced, integration cassette construction may become more efficient, either through a lower cost of synthesizing the DNA cassette or the use of PCR primers to provide the homology arms, as reported previously for E. coli [44]. The development of highly efficient DNA transformation and genome integration in Synechococcus sp.…”

Section: Discussionmentioning

confidence: 98%

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Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis

2016

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BackgroundSuccessful implementation of modified cyanobacteria as hosts for industrial applications requires the development of a cyanobacterial chassis. The cyanobacterium Synechococcus sp. PCC 7002 embodies key attributes for an industrial host, including a fast growth rate and high salt, light, and temperature tolerances. This study addresses key limitations in the advancement of Synechococcus sp. PCC 7002 as an industrial chassis.ResultsTools for genome integration were developed and characterized, including several putative neutral sites for genome integration. The minimum homology arm length for genome integration in Synechococcus sp. PCC 7002 was determined to be approximately 250 bp. Three fluorescent protein reporters (hGFP, Ypet, and mOrange) were characterized for gene expression, microscopy, and flow cytometry applications in Synechococcus sp. PCC 7002. Of these three proteins, the yellow fluorescent protein (Ypet) had the best optical properties for minimal interference with the native photosynthetic pigments and for detection using standard microscopy and flow cytometry optics. Twenty-five native promoters were characterized as tools for recombinant gene expression in Synechococcus sp. PCC 7002 based on previous RNA-seq results. This characterization included comparisons of protein and mRNA levels as well as expression under both continuous and diurnal light conditions. Promoters A2520 and A2579 were found to have strong expression in Synechococcus sp. PCC 7002 while promoters A1930, A1961, A2531, and A2813 had moderate expression. Promoters A2520 and A2813 showed more than twofold increases in gene expression under light conditions compared to dark, suggesting these promoters may be useful tools for engineering diurnal regulation.ConclusionsThe genome integration, fluorescent protein, and promoter tools developed in this study will help to advance Synechococcus sp. PCC 7002 as a cyanobacterial chassis. The long minimum homology arm length for Synechococcus sp. PCC 7002 genome integration indicates native exonuclease activity or a low efficiency of homologous recombination. Low correlation between transcript and protein levels in Synechococcus sp. PCC 7002 suggests that transcriptomic data are poor selection criteria for promoter tool development. Lastly, the conventional strategy of using promoters from photosynthetic operons as strong promoter tools is debunked, as promoters from hypothetical proteins (A2520 and A2579) were found to have much higher expression levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0584-6) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 98%

Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis

2016

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“…Red/ET-mediated recombination is a powerful homologous recombination system based on the function of either the Red operon of lambda phage or RecE/RecT from Rac phage [ 12 - 15 ]. To evaluate the cloning specificity of conventional Red/ET-mediated homologous recombination, we conducted a pilot experiment using a linear nonselective vector (NS-vector) and an artificially amplified V gene and mock DNA.…”

Section: Resultsmentioning

confidence: 99%

“…Red/ET-mediated recombination was carried out as described previously [ 15 ]. Vectors were linearized outside of the homology region by digestion with Eco RV.…”

Section: Methodsmentioning

confidence: 99%

Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

2011

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BackgroundMolecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.ResultsWe developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.ConclusionThe time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

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“…Introduction of the PCR-amplified V gene fragments into expression plasmids has been performed using traditional cut-and-paste DNA cloning techniques [5-9]. Recently, site-specific recombination and homologous recombination cloning techniques, which eliminate the use of restriction endonucleases and ligases, offer several advantages in the context of high-throughput procedures [10-14]. These methods, however, still require plasmid amplification in bacteria, followed by plasmid purification and verification of the insert.…”

Section: Introductionmentioning

confidence: 99%

Target-selective joint polymerase chain reaction: A robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells

2011

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BackgroundDuring the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies.ResultsWe have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days.ConclusionOur system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.

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Minimum Length of Homology Arms Required for Effective Red/ET Recombination

Cited by 8 publications

References 8 publications

Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis

Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis

Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

Target-selective joint polymerase chain reaction: A robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells

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