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Team:Goettingen/notebook wetlab/gfp
From 2014.igem.org
GFP team
A vial of specially modified GFP scaffold that could accommodate up to two short-peptide fragments was procured from Dr Tej V Pavoor from the university of Wisconsin-Madision, USA. Several amino-acid residues had been altered so that it could withstand the addition of peptide loops without compromising the fluorescence capacity significantly.
We intend to insert the peptide interacting with the membrane protein of the fungal pathogen to dummy loop 1 (which, along with dummy loop 2, are so called because of them just being present as a filler to be replaced) of the GFP scaffold. In our case it was IGP4, the peptide interacting with SSR1 from Candida glabarata.
The GFP scaffold without IGP4 will be used as a negative control, to rule out any interaction between the modified GFP scaffold and SSR1. Wild type Candida glabarata will be used for in vivo studies with a SSR1 knock out strain as negative control.
September 22nd, week 20
-PCR amplify Region I (RI) of GFP scaffold with primers that would exclude dummy loop 1 using PfuS PCR.
-PCR amplify Region II (RII) scaffold of GFP with BamHI restriction site PfuSPCR.
-Amplify IGP4 with left and right flanks that would overlap RI and RII regions of GFP PfuSPCR.
-Check the concentrations of RI, RII and IGP4.
Nanodrop
