Team:Tel-Hai/Notebook

iGEM Tel-Hai 2016

pSB1C3 - the backbone through which the parts should be sent
J04450 - the plasmid pSB1C3 with an added RFP gene (coding a red fluorescence protein)
K60800 - a plasmid containing a GFP gene

All plasmids were transformed into Top 10 E. Coli strain, using the heat shock method.
E. coli were spread onto LB agar plates with the antibiotic chloramphenicol and were incubated ON at 37o.

LB was inoculated with colonies of E.coli each transformed with one of the plasmids above that grew ON
Colonies were incubated at 37o ON

Extraction of plasmids from colonies using Miniprep kit and validation of presence of plasmid by running extraction product through agarose gel (1%) - fig 1.

Fig. 1 - Extraction of pSB1C3, J04450, K60800

Restriction of the plasmids pSB1C3 and J04450 with EcoRI and PstI. Incubation for 1 hour at 37°C
We ran the restriction products in agarose gel (1%)
Gel results indicated that that the plasmid J04450 was ok. With the plasmid pSB1C3 we did the procedure once again (fig 2)

Fig.2 - Restriction of the plasmids pSB1C3 and J04450 with EcoRI and PstI

We received the parts 1-4 from IDT
- part 1- contains the sequence of tGFP gene under EF1a promoter
- part 2- contains the sequence of CFTR exon 11, donor part
- part 3- contains the sequence of gRNA1 for CFTR ΔF508
- part 4- contains the sequence of gRNA2 for CFTR ΔF508
Restriction of the parts and the plasmid pSB1C3
Ligation of the parts to the plasmid backbone pSB1C3
All ligated plasmids were transformed into Top 10 E. Coli strain, using the heat shock method.
E. coli were spread onto LB agar plates with the antibiotic chloramphenicol and were incubated ON at 37o

Restriction of the plasmids pSB1C3 + parts 1-4 with EcoRI and PstI. Incubation for 1 hour at 37°C
We ran the restriction products in agarose gel (1%)
Gel results indicated that the ligation of the plasmid with part 4 was ok (fig 3)

Fig.3 - Restriction of the plasmids pSB1C3 + parts 1+4 with EcoRI and PstI
With all the other parts, we did the procedure again at 10-16.8.16 and the ligation worked for part 1 and part 3 and for part 2 it didn’t work (fig 4)

Fig.4 - Restriction of the plasmids j044540 + parts 1+2+3 with EcoRI and PstI

We received lung epithelial cells NCI-H 1650
The cells were thawed and seeded in a 6 well plate and incubated in a suitable incubator at 37o and 5% CO2

We ran the crosslink product in agarose gel (3 %)
The gel results indicates that there is a gel shift. The DNA runs through the gel in a different way compare to the negative control (pSB1C3).

Fig.5 - crosslink product. pSB1C3+CTB migration through agarose gel 3%. First well is the marker, Second well is only pSB1C3, Third well CTB only (for negative control), Fourth Well is pSB1C3+ CTB cross link showing a gel shift due to crosslinking.

we received a chimeric LTB protein with a DNA binding domain, dyed with FITC (green fluorescence)
we ran the protein in a polyacrylamide gel for verification of the protein size
the gel result indicate that the protein is at the correct size, ~60kD (fig 6)

Fig.6 - LTB+DBD dyed with FITC, in polyacrylamide gel.

we dyed a plasmid containing a GFP (pEGFPN3) with Hoechst stain (blue fluorescence)

pEGFPN3 plasmid
we purified the plasmid from the remaining dye using a Miniprep kit
we incubated the plasmid + Hoechst with the LTBD + FITC for 45 min. at 37o
NCI-H 1650 cells were washed and prepared for incubation with plasmid and protein complex and prepared for FACS
FACS analysis below (fig 7) shows that the protein the cells but we were not able to detect the blue Hoechst staining

Fig.7 - FACS results

we incubated the plasmid + Hoechst with the CTBD + FITC for 1 hour at 37o
Cell medium was added to the complex to achieve 200µl and the whole reaction was added to the cells.
Cells were incubated in a suitable incubator at 37o and 5% CO2 wrapped in aluminum foil for 5 hours
Cells were pictured using a confocal microscope
The picture shows (fig 8) the green staining of FITC inside the cells. Looking at the Hoechst staining we received a lot of background and assumed that the DNA degraded

Fig.8 - confucal results. Green staining of epithelial cells (NCI-H1650) by LTBD stained with FITC. LTBD binds specifically GM1 on epithelial cells and enters by endocytosis

we linearized the plasmid pEGFPN3 using EcoR1, 1 hour at 37o
we incubated the linearized plasmid with increased concentrations of the protein- 30,60,90,150,300,600 ng, and with a CTB protein without a DNA binding domain, for 45 min. at 37o
we ran the reactions in agarose gel (1%)
gel results (fig 9) indicate that the optimal concentration of protein that binds 400ng of DNA is 300ng

Fig.9 - Binding pSB1C3 plasmid in constant concentration (400ng) to LTBD. gel results indicate that the optimal concentration of protein that binds 400ng of DNA is 300ng. *LTBD - Heat-Labile Toxin (LT), an analog to CT