NASC Stock Detail Page
NASC ID: N2105633
Name: pART7::d-FlincG vector
ABRC stock number: CS2105633
Description: The d-FlincG (fluorescence indicator of cGMP) biosensor is based on cpEGFP (circular permutated EGFP), C-terminally fused to the in-tandem regulatory domain of type-I PKG. The N-terminal dimerization domain of the PKG was removed, resulting in d-FlincG with an increased dynamic range (Nausch et al., 2008). In order to express d-FlincG in plants, PKG was subcloned in pART7 under the control of the 35S constitutive promoter. pRSET-A::d-FlincG was kindly provided by Drs Naush and Dostmann (College of Medicine, Department of Pharmacology, University of Vermont) carrying a copy of PKGI with cpEGFP inserted in the middle, and carrying a stop codon. pRSET-A:: d-FlincG was cut with BamHI, and Klenow was applied in the presence of dGTP and dATP. pART7 was cut with XhoI, and Klenow was applied in the presence of dTTP and dCTP. The pART7 and pRSET-A::d-FlincG were subsequently cut with EcoRI, and the released fragments of interest were ligated, with XhoI and BamHI providing compatible cohesive ends. The catalytic subunit of PKGI, which is not translated, was removed by cutting pART7::d-FlincG with the two compatible end enzymes AvrII and XbaI, followed by self-ligation. The NotI fragment of pART7::d-FlincG containing the 35S::d-FlincG construct was subcloned in pGREEN 0229. Three-week-old Arabidopsis plants were transformed using the flower-dipping method according to Clough and Bent (1998). Seeds of these plants were collected and grown on soil for 10 days, and were treated with the herbicide BASTA 7 . After 1 week, BASTA -resistant seedlingswere selected and screened for their GFP expression.
Donation Date: 2015-01-23
| Donated by: | University of Bristol Jean Charles Isner |
Part of : Isner Collection
Stock type: individual line
Material type: plasmid dna
Comment: Related stocks: There is a seed stock relating to this line ( N2105635.)
| Status | Price (£) |
|---|---|
| Available | £11.00 |
Germplasm Info
Phenotype
pART7::delta-Flinc
References
Isner, J.C. & Maathuis, F.J. 2011. Measurement of cellular cGMP in plant cells and tissues using the endogenous fluorescent reporter FlincG. The Plant Journal 26(2):329-34. PubMed ID: 21223396.