pMAL-EFSep
(Plasmid #34627)

• Depositing Labs: Jesse Rinehart, Dieter Söll
• Publication: Park et al Science. 2011 Aug 26;333(6046):1151-4.

Backbone

• Vector backbone: pMAL-c2x
• Backbone manufacturer: NEB
• Backbone size w/o insert (bp): 6700
• 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: EF-Sep
• Alt name: tufB
• Alt name: EF-Tu
• Species: E. coli
• Insert Size (bp): 1184
• Mutation: His67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, Asn274 to Trp, and Val 351 Leu
• Entrez Gene: tufB (a.k.a. B21_03809)
• Tags / Fusion Proteins:
  • 6xHIS (C terminal on insert)
  • MBP (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRI (unknown if destroyed)
• 3′ cloning site: Pst (unknown if destroyed)
• 5′ sequencing primer: MBP_F
• 3′ sequencing primer: pBAD_rev

Terms and Licenses

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

Depositor Comments

To construct pMAL-EFSep the gene encoding EF-Sep, was cloned between the NdeI and BamHI sites in the pET20b plasmid (Novaven) to
add a C-terminal His6 tag. This fusion construct was then PCR-amplified using primers adding MfeI and PstI restriction sites. The PCR product was cloned in-frame between EcoRI and PstI in pMALc2x (New England Biolabs) to add an N-terminal maltose binding protein (MBP) tag.

To prevent possible enzymatic dephosphorylation of O-phospho-L-serine (Sep) in vivo, the gene encoding phosphoserine phosphatase (serB), which is catalyzing the last step in serine biosynthesis, was deleted from Escherichia coli strains Top10 (Top10∆serB - Addgene #34928) and BL21 (BL21∆serB - Addgene #34929). These strains are required hosts when using this plasmid.