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

• Purpose: a single-wavelength intracellular glutamate sensor constructed from E. coli Gltl and cpGFP. For bacterial cytoplasmic expression
• Depositing Lab: Loren Looger
• Publication: Marvin et al Nat Methods. 2013 Feb;10(2):162-70. doi: 10.1038/nmeth.2333. Epub 2013 Jan 13.

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, 100 μg/mL
• 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

Resource Information

• Supplemental Documents:
  • ybeJ253.L1LV.L2NP.pRSET.pdf
  • ybeJ253.L1LV.L2NP.pRSET.sbd
  • ybeJ253.L1LV.L2NP.pRSET.gb
• A portion of this plasmid was derived from a plasmid made by: E. coli glutamate-binding protein (gltI) from FLIP-E (Okumoto, et al. 2005, PNAS, PMID: 15939876) and cpGFP146 variant from EcMBP165-cpGFP.PPYF.pRSET (Addgene plasmid #33372)

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

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