PurposeExpression of Spectrin-cpstFRET to measure mechanical stress and report multidimensional stress fields
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||61109||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4700
- Total vector size (bp) 12863
Modifications to backboneBased on Venus and Cerulean, we constructed circularly permutated cpVenus and cpCerulean. For cpVenus, we first subcloned the gene fragments of 175Gly–239Lys from Venus into pET-52b (+) vector with BamHI and EcoRI. Then we subcloned the gene fragments of 1Met–174Asp and connected them to the C-terminal of the first fragment (supplementary material Fig. S1). Five glycines were added between them to give flexibility with EcoRI and SacI. We generated cpCerulean similarly except that a different primer was used for fragment 175Gly–239Lys, which was 59- GGTACCAGGATCCATGGGCAGCGTGCAGC-39 with BamHI. For eukaryotic cell expression, we subcoloned cpVenus and cpCerulean into pEGFP vector with EGFP removed beforehand, and with Nhe1 for cpCerulean and with NheI for cpVenus. pET-cpstFRET was created by connecting cpVenus to the C-terminal of cpCerulean in pET-52b(+) vector with BglII and SacI. Fore eukaryotic cell expression of cpstFRET, we subcloned it into pEGFP vector with NheI and ApaI. To create chimeric gene constructs of α-spectrin, we subcloned the gene encoding spectrin into pEGFP-C1 vector with AgeI and SacII from which the EGFP gene was removed beforehand. cpst-FRET was inserted into spectrin at amino acid position 1200, which located in the linker domain between the 10th and 11th spectrin repeat domains by restriction sites SalI and NotI.
Vector typeMammalian Expression
Selectable markersNeomycin (select with G418)
Growth in Bacteria
- Promoter CMV
- Cloning method Restriction Enzyme
- 5′ cloning site AgeI (not destroyed)
- 3′ cloning site SacII (not destroyed)
- 5′ sequencing primer CMV Forward
- 3′ sequencing primer SV40 poly A Reverse (Common Sequencing Primers)
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:Spectrin-M-cpstFRET was a gift from Fred Sachs (Addgene plasmid # 61109 ; http://n2t.net/addgene:61109 ; RRID:Addgene_61109)
For your References section:Orientation-based FRET sensor for real-time imaging of cellular forces. Meng F, Sachs F. J Cell Sci. 2012 Feb 1;125(Pt 3):743-50. doi: 10.1242/jcs.093104. 10.1242/jcs.093104 PubMed 22389408