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 λ-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.

  • Source
    • "Although much success has been achieved regarding isoprene biosynthesis, many problems remain to be solved, such as the intermediate imbalance resulting from heterologous over-expression of so many non-native genes in the host[12]. To overcome these hurdles, one approach is to employ a chromosome integration method to reduce the burden of cell growth resulting from the over-expression of heterologous genes[13,14]. Recent advances in synthetic biology and metabolic engineering have made it possible to construct a new pathway to replace the native pathway by optimizing and assembling different sources of the enzymes. "

    Preview · Article · Dec 2016 · BMC Biotechnology
  • Source
    • "LEDs were powered using an Arduino duemilanove microcontroller (Arduino) and controlled in concert with the filter wheel using Micromanager. Chromosomal constitutive mRFP1 mRFP1 (BBa_E1010) driven by the lambda phage PR promoter (BBa_R0051) was cloned into the " landing pad " region of pTKS/CS that included a tetracycline resistance cassette (Kuhlman & Cox, 2010). Linear DNA containing the landing pad region was amplified using primers to add 50 bp of homology to the coding region of theSabri et al, 2013) on each end of the landing pad. "

    Full-text · Article · Jan 2016 · Molecular Systems Biology
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jun 2015 · Metabolic Engineering
Show more