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Biosensor libraries harness large classes of binding domains for allosteric transcription regulators

Javier F. Juárez, Begoña Lecube-Azpeitia, Stuart L. Brown, George M. Church
doi: https://doi.org/10.1101/193029
Javier F. Juárez
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Begoña Lecube-Azpeitia
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Stuart L. Brown
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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George M. Church
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Abstract

Bacteria’s ability to specifically sense small molecules in their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource. While many transcription factors (TFs) mediating these processes have been studied, only a handful has been leveraged for molecular biology applications. To expand this panel of biotechnologically important sensors here we present a strategy for the construction and testing of chimeric TF libraries, based on the fusion of highly soluble periplasmic binding proteins (PBPs) with DNA-binding domains (DBDs). We validated this strategy by constructing and functionally testing two unique sense-and-response regulators for benzoate, an environmentally and industrially relevant metabolite. This work will enable the development of tailored biosensors for synthetic regulatory circuits.

  • ABBREVIATION LIST

    3D
    Three-dimensional
    ApmR
    Resistance to ampicillin/carbenicillin
    aTc
    Anhydrotetracycline
    CmR
    Resistance to chloramphenicol
    CRISPR
    Clustered Regularly Interspaced Short Palindromic Repeats
    Ct
    C-terminal
    DBD
    DNA-Binding Domain
    dCas9
    Defective Cas9 protein, able to bind DNA and RNA but lacking nuclease activity
    eLCR
    enhanced LCR
    FACS
    Fluorescence-Associated Cell Sorting
    GGBP
    glucose-binding protein (E. coli)
    GmR
    Resistance to gentamycin
    IPTG
    Isopropyl β-D-1-thiogalactopyranoside
    KmR
    Resistance to kanamycin
    LCR
    Ligase Chain Reaction
    LNK
    linker
    nSP
    SBD that does Not contain its Signal Peptide
    Nt
    n-terminal
    PBP
    Periplasmic Binding Protein
    PBS
    Phosphate Buffered Saline
    RBS
    Ribosome Binding Site
    SBD
    Substrate-Binding Domain
    OD
    Oligomerization Domain
    PID
    Percentage of Identity
    SP
    Signal Peptide
    TALENs
    Transcription activator-like effector nuclease
    TF
    Transcription factor
  • Copyright 
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    Posted September 23, 2017.
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    Biosensor libraries harness large classes of binding domains for allosteric transcription regulators
    Javier F. Juárez, Begoña Lecube-Azpeitia, Stuart L. Brown, George M. Church
    bioRxiv 193029; doi: https://doi.org/10.1101/193029
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    Biosensor libraries harness large classes of binding domains for allosteric transcription regulators
    Javier F. Juárez, Begoña Lecube-Azpeitia, Stuart L. Brown, George M. Church
    bioRxiv 193029; doi: https://doi.org/10.1101/193029

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