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pRSET.GltI253-cpGFP.L1LV/L2NP
(Plasmid #41733)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 41733 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pRSET-A
  • Backbone manufacturer
    Invitrogen
  • Backbone size w/o insert (bp) 2897
  • Total vector size (bp) 4432
  • Vector type
    Bacterial Expression ; Glutamate Biosensor

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    GltI based Glutamate Biosensor
  • Alt name
    iGluSnFR
  • Alt name
    gltI/ybeJ
  • Alt name
    cpGFP146
  • Species
    Synthetic; E. coli
  • Insert Size (bp)
    1573
  • Mutation
    cpGFP146 variant from EcMBP165-cpGFP.PPYF.pRSET inserted between AA253-254 of E. coli gltI/ybeJ
  • Promoter T7
  • Tags / Fusion Proteins
    • 6xHis (N terminal on backbone)
    • T7 epitope (N terminal on backbone)
    • Xpress tag (N terminal on backbone)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site BamHI (not destroyed)
  • 3′ cloning site EcoRI (not destroyed)
  • 5′ sequencing primer T7
  • 3′ sequencing primer T7-term
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

Biosensor iGluSnFRa is a single-wavelength glutamate sensor constructed from E. coli GltI and cpGFP. iGluSnFR is bright and photostable, with 4.5 (ΔF/F)max in vitro, under both 1- and 2-photon illumination.

pRSET.GltI253-cpGFP.L1LV/L2NP was constructed by subcloning the gene for E. coli glutamate-binding protein (gltI) from a previous glutamate sensor, FLIP-E (Okumoto, et al. 2005, PNAS, PMID: 15939876), into the pRSET-A vector (Invitrogen) with a BamHI site encoding Gly-Ser at the 5’-end and an EcoRI site included after the stop codon. The GltI-cpGFP insertion variants were constructed by overlap PCR using the wild-type gltI sequence and the cpGFP146 variant from EcMBP165-cpGFP.PPYF.pRSET (Addgene plasmid #33372). The linker was selected by high-throughput screening of site-directed mutagenesis-generated linker variants. Detailed amino acid sequences are provided in the supplementary documents available above. More information on senor engineering and characterization can be found in the associated article (see below).

Alternate plasmid names: ybej253.L1LV.L2NP.pRSET; pRSET.iGluSnFR

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pRSET.GltI253-cpGFP.L1LV/L2NP was a gift from Loren Looger (Addgene plasmid # 41733)
  • For your References section:

    An optimized fluorescent probe for visualizing glutamate neurotransmission. Marvin JS, Borghuis BG, Tian L, Cichon J, Harnett MT, Akerboom J, Gordus A, Renninger SL, Chen TW, Bargmann CI, Orger MB, Schreiter ER, Demb JB, Gan WB, Hires SA, Looger LL. Nat Methods. 2013 Feb;10(2):162-70. doi: 10.1038/nmeth.2333. Epub 2013 Jan 13. 10.1038/nmeth.2333 PubMed 23314171