EcMBP165-cpGFP.PPYF.pRSET
(Plasmid #33372)

• Purpose: a single-wavelength maltose sensor constructed from E. coli MalE and cpGFP
• Depositing Lab: Loren Looger
• Publication: Marvin et al Proteins. 2011 Nov;79(11):3025-36. doi: 10.1002/prot.23118.

Backbone

• Vector backbone: pRSET-A
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 2900
• Vector type: Bacterial Expression

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: MBP based Maltose Biosensor
• Species: E. coli
• Mutation: cpGFP146 variant from GCaMP2 inserted between AA165-166 of MBP
• Promoter: T7
• Tags / Fusion Proteins:
  • 6xHIS (N terminal on backbone)
  • 6xHIS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: T7 forward

Terms and Licenses

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

Depositor Comments

The MBP-cpGFP insertion variant was constructed by overlap PCR using the wild-type MBP sequence from the pMalE-p4E vector (New England Biolabs) and the cpGFP146 variant from GCaMP2 into pRSET-A between BamHI and EcoRI sites. The N-terminal periplasmic leader sequence of MBP was excluded, and a BamHI site (encoding Gly-Ser) was included at the 5′-end. At the C-terminus, an additional His6Gly tag was included after the terminus of MBP (a cloning oversight). An EcoRI site was included after the stop codon.