|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||70188||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Backbone manufacturerFagan and Fairweather 2009
- Backbone size w/o insert (bp) 7216
- Total vector size (bp) 9178
Modifications to backboneReplaced gusA from original pRPF185 with amyEopt
Vector typeBacterial Expression, Synthetic Biology
Growth in Bacteria
Growth instructionsFor E.coli, use Cm at 20ug/mL for growth on solid media, but 10ug/mL for growth in liquid medium. For C. difficile use thiamphenicol at 15ug/mL.
Copy numberHigh Copy
Alt namesecreted alpha-amylase
Insert Size (bp)1962
- Promoter Ptet
- Cloning method Restriction Enzyme
- 5′ cloning site SacI (not destroyed)
- 3′ cloning site BamHI (not destroyed)
- 5′ sequencing primer NF_1323 (CTGGACTTCATGAAAAACTAAAAAAAATATTG)
- 3′ sequencing primer NF_794 (CACCGACGAGCAAGGCAAGACCG) (Common Sequencing Primers)
Please note: the depositing lab has determined that a particular IS element present in E. coli strains such as DH5alpha and its derivatives has a high propensity to insert into this plasmid. The depositing lab recommends isolating the plasmid DNA and transforming into a strain similar to MC1061.
Publication describing the backbone vector pRPF185 as well as the sequencing primers:
Clostridium difficile has two parallel and essential Sec secretion systems. Fagan RP, Fairweather NF. J Biol Chem. 2011 Aug 5;286(31):27483-93. doi: 10.1074/jbc.M111.263889. Epub 2011 Jun 9.
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:pWKS1583 was a gift from Wiep Klaas Smits (Addgene plasmid # 70188 ; http://n2t.net/addgene:70188 ; RRID:Addgene_70188)
For your References section:The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile. Oliveira Paiva AM, Friggen AH, Hossein-Javaheri S, Smits WK. ACS Synth Biol. 2016 Jun 23. 10.1021/acssynbio.6b00104 PubMed 27333161