Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38, e92

Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544-1014 USA.
Nucleic Acids Research (Impact Factor: 9.11). 04/2010; 38(6):e92. DOI: 10.1093/nar/gkp1193
Source: PubMed


We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses lambda-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol.

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    • "However, we were able to achieve 59%, 35%, and 14% efficiency for inserting 3 kb, 5 kb, and 8 kb sequences, respectively. This increased efficiency was possibly attributed to DSB generated recombination stimulation effect (Fig. 2e) (Kuhlman and Cox, 2010). "
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    ABSTRACT: Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0g/L β-carotene in fed-batch fermentation. Copyright © 2015. Published by Elsevier Inc.
    Metabolic Engineering 06/2015; 31. DOI:10.1016/j.ymben.2015.06.006 · 6.77 Impact Factor
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    • "This counter-selection was deemed to be the most efficient hence the use of MG1655 rather than BW25113, which has a Δrha genotype [51] and therefore can neither import nor metabolize rhamnose. Using these templates, the counter-selectable cassette was inserted into the cyd and nuo operons at the insertion sites for the mMaple gene by λ-red recombination with λ-red plasmid pTKRED as previously described [55]. "
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    ABSTRACT: Oxidative phosphorylation (OXPHOS) is the essential process which oxidizes reducing equivalents obtained from nutrient breakdown to energize transmembrane ion translocation. The electrochemical gradient is coupled to generation of the cellular fuel ATP. OXPHOS requires multiple membrane-integrated protein complexes, usually located in the cytoplasmic membrane in prokaryotes, or in the inner mitochondrial membrane in eukaryotes. To determine the distribution and dynamics of OXPHOS components in an intact membrane, we used real-time multicolor single-molecule fluorescence imaging and super-resolution particle tracking on live cells of Escherichia coli expressing combinations of chromosomally-encoded fluorescent protein fusions to five essential OXPHOS complexes: cytochromes bd-I and bo terminal oxidases, FoF1ATPase, NADH dehydrogenase and succinate:fumarate oxidoreductase. In all cases we observed distinct fluorescent spots in the cytoplasmic membrane whose width was 100-200 nm larger than that of single surface-immobilized fluorescent protein, each containing as few as ~10 molecules up to several hundred, estimated using photophysical analysis and photoactivated localization microscopy (PALM). Bayesian inference indicates ~57% of spots are mobile, diffusing at ~0.007 μm2 s1, while the remaining spots have confined mobility in domains of ~100 nm diameter. Spots associated with different complexes show no intrinsic co-localization. Fluorescence recovery after photobleaching (FRAP) experiments indicate rapid long-range diffusion of the ubiquinone electron-carrier which could be an important limiting factor in electron transport. OXPHOS complexes are compartmentalized as assemblages of distinct, dynamic islands. Both electron transport and the proton circuit are delocalized, indicating an OXPHOS strategy very different to that found in mitochondria.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 06/2014; 1837(6):811-824. DOI:10.1016/j.bbabio.2014.01.020 · 5.35 Impact Factor
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    • "A strategy that can exclusively yield efficient site-specific integration into safe locations in the target genome would be ideal. However, while homologous recombination provides excellent specificity in integration sites, it occurs at too low frequency to be optimal for genetic engineering in multicellular organisms (Kuhlman and Cox 2010). Site-specific DNA recombination systems are derived from prokaryotes and unicellular yeasts, and among them, bacterial viruses provide a repertoire of recombinational systems, a number of which have been exploited to facilitate efficient DNA exchange in human cells. "
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    ABSTRACT: Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.
    Applied Microbiology and Biotechnology 01/2014; 98(7). DOI:10.1007/s00253-014-5512-2 · 3.34 Impact Factor
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