Team:Colombia Uniandes/ChimiJournal

Hands at work!

Here you will find the overall progression of our work at the laboratory designing Chimi.

13th June 2013

The first thing we did was to extract the plasmids from the iGEM plaque.

1. From the 2013 kit, Plate 1, Well 19 – o
2. From the 2013 kit, Plate 1, Well 2 – i
3. From the 2013 kit, Plate 3, Well 17 – c

The iGEM parts were taken in order to perform an electroporation. For this, we use:

• 20 µ of miliQ water (ultra pure). Find the plate and stick the tip with water into the well, perforating the aluminum.
• Resuspend the well’s content by gentle pipetting.
• When the water has a dark red color, transfer it to a PCR eppendorf and put on ice.

15th June 2013

We stung the transformant colonies.

18th June 2013

We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.

This are the overall steps:

1. Harvest cells.
2. Resuspend cells.
3. Cell lysis.
4. Neutralization.

Spin method:

5. Prepare column.
6. Load cleared lysate.
7. Wash column with wash solution 1.
8. Was column with wash solution 2.
9. Centrifuge.
10. Elute DNA.

We did a confirmation Gel 100 V x 30 min. -> It showed 1 bond in the first two wells corresponding to Nal1 and Nal2. We succesfully extracted the Nal plasmids!

June 21st, 2013

Harja et al., “Bust n’ Grab” Protocol for Yeast Genomic DNA Extraction

1. 5 mL of overnight culture of S. cerevisiae (in BHI medium) were centrifuged at 8500 rpm for 5 min. Discard de supernatant.
2. 500 µL of Harja lysis buffer were added to each tube.
3. Place 2 min at -20 °C, 1 min in water bath at 90 °C and repeat.
4. Vortex 30 s.
5. Add 500 µL of chloroform, vortex 2 min and centrifuge 3 min at 8500 rpm.
6. Transfer the upper aqueous phase to a tube with 800 µL of chilled 100% ethanol and mix by inversion.
7. Incubate for 5 min at room temperature or at 30 °C.
8. Centrifuge for 5 min, 8500 rpm, and discard supernatant.
9. Wash the pellet with 500 µL of ethanol (100%) by vortex. Repeat step 9.
10. Dry pellets at room temperature or at 60 °C.
11. Resuspend in 40 µL miliQ water.

June 26, 2013

• We made competent yeast following the procedure mentioned before.
• We also made our first PCRs! We used primers 6 & 1 (A) and 34 & 9 (B) to extract VP16 and GCR from the Nal1 plasmid.
• Confirmation gel (2013-06-26 19 hr 16 min.jpg & 2013-06-26 19hr 15 min.jpg) with wells:
  1. Ladder
  2. PCR A
  3. PCR B
  4. Miniprep for Nal. 1

A = VP16
B = GCR

June 27, 2013

We repeated the PCR for A and used lambda phage DNA for carrier DNA. We also extracted yeast genome using a modified Harju “Bust n’ Grab” protocol using three parallel methods:

• Method “H” used the regular lysis buffer.
• Method “C” used the following lysis buffer: 3% Triton, 100 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaAc.
• Method “O” used the following lysis buffer: 2% triton, 1& SDS, 10 mM tris-HCl, 1 mM EDTA, 100 mM LiCl.

Conformation gel was run with wells:

1. Ladder
2. PCR A (repeated)
3. Carrier lambda PCR
4. Method C
5. Method H
6. Method O

We then ran a fusion PCR with GAL4 and VP16 (A and B). These were the PCR conditions:

• 1st PCR --> 2-step PCR
  Cycle steps
  1. Initial denaturation (98 °C, 30 s)
  2. 15 cycles (98 °C, 10 s; 72 °C, 35 s)
  3. Final extension (72 °C, 5 min)
  4. Hold (4 °C, indefinite time)
• 2nd PCR --> 2-step PCR (add primers)
  Cycle steps
  1. Initial denaturation (98 °C, 30 s)
  2. 35 cycles (98 °C, 10 s; 72 °C, 35 s)
  3. Final extension (72 °C, 10 min)
  4. Hold (4 °C, indefinite time)

June 28th, 2013

We extracted pBap2, Gal4 and pGal1 (C, D and E) from the genome of S. cerevisiae. We ran a gel with the following wells: WM, AB, C, D, E.

July 2nd, 2013

We ran out of yeast genome, so today we had to extract it again! We used two different solutions:

• Solution I: Glucose 50 mM, EDTA pH 8 10 mM, Tris-HCl pH 8 25 mM (esterilized)
• Solution II: NaOH 0.2 N, SPS 1%

July 5th, 2013

We performed yeast genome extraction using 4 methods.
General guidelines from the GenElute DNA Kit from Sigma-Aldrich.

• A: Zymolyase for ½ h at 37 °C
• B: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 55 °C
• C: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 37 °C
• D: Normal protocol


We ran a gel in this order: WM, A, B, C, D. (Positive for B, A, C. Brighter band in C.)
We performed PCRs for BAP2, GAL4, yeast terminator and pGAL1. These were the conditions:
PCR fusion 2 steps*, with process 3 at 72 °C for 45 s

*
1. 98 °C, 1 min
2. 98 °C, 10 s
3. 72 °C, 45 s

Steps 2 and 3 x 35

4. 72 °C, 10 min
5. 4 °C

July 9th, 2013

Today we ran PCRs for pGAL1 and mCherry. Now that we have several parts, we must have a clear understanding of our notation!

<il>pBap2 = C
• GAL4 = D
• VP16 = A
• GCR = B
• TER = F
• VP16 + GCR = AB
• pBAP2 + GAL4 = CD
• AB + TER = ABF

July 18th, 2013

Today we ran PCRs for the following fusions: F1 = TER-GCR, with primers 31 & 35; F2 = TER – mCherry, with primers 31 & 33. We also amplified E = pGAL1, using primers 13 & 15.

July 19th, 2013

Today we amplified F2 that we obtained yesterday.

July 22nd, 2013

Today we tried to fuse C-D using primers 19 and 5, TER-GCR using primers 31 and 35 and TER-mCherry using primers 31 and 33. After 15 PCR cycles, we added the primers.

July 24th, 2013

Today we extracted the terminator from the yeast genome (F1 and F2) and pGAL1 from miniprep (E). We also did PCRs to fuse again our big parts: pBAP2 – GAL4 – VP16 – GCR (CD-AB) (primers 19 & 34) and pGAL1 – mCherry (E-G) (primers 32 & 15).
At the moment, this is our progress!:




August 5th, 2013

Things are advancing at a fast pace! Today we did miniprep to obtain PUC19!

August 6th, 2013

After doing a confirmation gel, C and CD are no longer with us! So we need to repeat them!

August 16th, 2013

Still no C nor D, so we did PCRs again with different methods to obtain C1, C2, D1 and D2.

September 3rd, 2013

Today we confirmed what we will be sending for the regional competition!
qBlocks (200 ng)

• Terminator (114486764) (F1 & F2)
• 3X UAS-pGAL1 (114486765) (E2)
• 6X UAS-pGAL1 (114486766) (E3)
• 9X UAS-pGAL1 (114486767) (E4)

Final volume: (40 µL)
Final concentration: 5 ng/µL

<p>In our quest to obtain again all our parts, we performed the following fusion PCRs: ABF1 (primers 1 & 31), GF2 (primers 17 & 31), E1GF2 (primers 15 & 31), E2G (primers 15 & 13), E3G (primers 15 & 13), E4G (primers 15 & 13). </p>

TOPO transformation 1 µL wáter from the kit 1 µL salt solution 2 µL plasmid EG 1 µL TOPO vector 5 minute room temperatura. Transform on ice. TOPO was transformed and backbone was left at 37 °C. PCRs were taken and left at -30 °C September 4 AB1F1, AB2F1 AB4F1 GF2 E2G E3G E4G E1GF2 September 15 Ligation in pGEM-T-Easy September 20

Backbone digestion