|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||34627||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 6700
Vector typeBacterial Expression
Growth in Bacteria
Copy numberHigh Copy
Insert Size (bp)1184
MutationHis67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, Asn274 to Trp, and Val 351 Leu
Entrez GenetufB (a.k.a. B21_03809)
/ Fusion Proteins
- 6xHIS (C terminal on insert)
- MBP (N terminal on backbone)
- 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 (Common Sequencing Primers)
Terms and Licenses
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.
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:pMAL-EFSep was a gift from Jesse Rinehart & Dieter Söll (Addgene plasmid # 34627 ; http://n2t.net/addgene:34627 ; RRID:Addgene_34627)
For your References section:Expanding the genetic code of Escherichia coli with phosphoserine. Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ, Rinehart J, Soll D. Science. 2011 Aug 26;333(6046):1151-4. 10.1126/science.1207203 PubMed 21868676