Difference between revisions of "Team:Tel-Hai/Notebook"

30.6.16

Extracting the plasmids from the iGEM kit:

1. pSB1C3 - the backbone through which the parts should be sent
2. J04450 - the plasmid pSB1C3 with an added RFP gene (coding a red fluorescence protein)
3. 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.

6.7.16

• 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

7.7.16

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

  

13.7.16

• 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)

  

3.8.16

• 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

7.8.16

• Extraction of plasmids from colonies that grew using Miniprep kit

8.8.16

• 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)
  

  

• 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)
  

  

17-18.8.16

• We received the purified CTB protein from SIGMA-ALDRICH
• The CTB was crossed linked to the plasmid pSB1C3 using a chemical linker

29.8.16

• 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

31.8.16

• 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).

  

11.9.16

• 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)

  

20.9.16

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

  

• 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
  

  

Preparing cells for confocal-

• We seeded cells in 24 well confocal plates for the experiment the next day

21.9.16

• 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
  

  

9.10.16

Binding plasmid in constant concentration (400ng) to CTB + DNA binding domain

• 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