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:

PCR Product	ng/μl
RI	373.6
RII	326.1
IGP4	89.7

-Verify PCR amplified products on 1% agarose gel to check if the amplicon is of the right size, followed by purification.

September 29th, week 21

-Perform an overlap extension PCR (Fusion PCR) to join our fragments RI, IGP4, and RII in the ratio 1:1.5:1 respectively.
-On a preparative gel, load all the PCR products, run the gel, excise the fragments of the right size and purify.
-In parallel, GFP scaffold (without IGP4) was also processed in the same way.

Preparative Agarose Gel (1%):

Nanodrop:

Gel Extracted	ng/μl
GFP	111.5
IGP4-GFP	130.6

October 6th, week 22

-Measure DNA concentration of pGP172 → 410 ng/µl.
-Use 5000 ng of pGP172 Vector for restriction. 13µl Vector, 1 µl H2O, 4µl buffer (Tango), 2 µl Sma1 and incubate for 2 h at 30°C.
Then add 2 µl of BamH1, 4µl buffer (Tango), and incubate for 2h more at 37°C. dephosphorylation with SAP for additional 30 minutes.
-Stop restriction digestion by purifying the mixture using PCR purification kit and check the fragments on a gel.

Agarose Gel (1%):

Nanodrop:

PCR Purified	ng/μl
pGP172	63
GFP	33.9
IGP4-GFP	86.7

-Inserts GFP and IGP4-GFP are ligated to vector pGP172 over night at 16 °C
-The Ligation mixture is transformed into E.coli DH5alpha competent cells and plated on LB+Amp plates, incubated at 37 °C overnight.
-Pick 8 colonies from each plate and run a Colony PCR. Check the results on 1% agarose gel, inoculate the positive colonies and incubate them overnight at 37 °C for plasmid extraction.

⇒ Bands 4 and 5 from IGP4-GFP colonies are positive
-Extract plasmids from the positive colonies, and check their concentration.

Nanodrop:

Gel Extracted	ng/μl
GFP	4255
IGP4-GFP	1533.3

October 13th, week 23

-1μl of extracted plasmid is transformed into E.coli BL21 Blue competent cells and plated on LB+Amp plates, incubated at 37 °C overnight.
-Pick 8 colonies for inoculating 4ml LB+Amp tubes and incubate them overnight at 37 °C.
- Test expression, protein extraction, protein purification and in vivo studies are under progress.