Team:Colombia Uniandes/Journal

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 picked 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

Harju 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 Harju 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 tried extracting 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

The genome extraction still isn't working! :(

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)

July 2nd, 2013

Still trying to successfully extract the yeast genome! This time we tried an alternate method where we used two different solutions to break the cell wall:

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

The rest of the protocol was taken from GenElute DNA Kit from Sigma-Aldrich.

July 3rd, 2013

The genome extraction was better, but it's mostly degraded DNA! We still have to improve the protocol.

July 5th, 2013

We're still improving our genome extraction protocol. This time we're trying 4 variations to break the cell wall
We're varying the incubation of both solutions, the one that comes with the kit (Proteinase K + Lysis buffer) and the zymolyase solution we previously used. The four variations were as follows.

• 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: Regular GenElute Genome extraction protocol

The rest of the steps were done following the instructions from the GenElute Genome extraction protocol.


We ran a gel in this order: WM, A, B, C, D.
Both B and C gave results, with C giving better yields! We're keeping the C protocol and we're happy we can start extracting parts from the yeast genome!






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!

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.

August 17th,2013

We're now starting with the reporter construct's PCRs. We got the synthetised parts (E2, E3, E4 and F (the terminator)) which means we need to start fusing them. First off, we'll fuse the terminator with the reporter, since it's common to all reporters.

August 19th,2013

Today we'll perform the fusion between FG and the four Es.

August 20th,2013

The fusion didn't work out, so we reamplified FG and tried again.

August 30th, 2013

We finally have the E*GF fusions complete! We did the digestion for the E*GF parts and the pSB1C3 plasmid as follows:

• Buffer 5x: 5 uL
• SpeI: 1 uL
• XbaI: 1 uL
• DNA: 10 uL
• Water: 33 uL

They were placed for two hours at 37°C and then were inactivated at 80°C for 20 minutes. Then we added alkaline phosphatase for 1 hour at 37°C.

Then, we did the ligation protocol as follows:

• Buffer 5x: 2 uL
• plasmid: 3 uL
• insert: 9 uL
• ligase: 1 uL
• Water: 5 uL

We then transformed them. Tomorrow we'll confirm the transformation by miniprep kit.

August 31st

After transforming them we got these minipreps




Here we can see all four E*GF parts (the four to the left of the weight marker), some with more than one plasmid conformation.

September 2nd, 2013

We contransformed the E*GF parts with the NAL plasmid in order to validate their function. Once again we did electrocompetent cells and transformed immediately.

September 3rd, 2013

We did the PCR confirmation for the parts. The bands aren't very bright but it's enough for us! We'll run the validation experiments tomorrow!

Wells are as follows: E1FG - E1FG(2) - E2FG - E2FG(2) - Weight marker - E3FG - E3FG(2) - E4FG - E4FG(2). Something strange happened with E3FG(2)




September 4th, 2013

Everything is ready for the validation experiments. We're both nervous and excited!

We inoculated cells with dexamethasone. The original syringe had a concentration of 8mg/2mL. We obtained a total initial volume of 3.189*10^-2 µL, so we had to dilute first the dexamethasone: 9900 µL water + 100 µL dexamethasone.

Induction experiment ON: 5 mL LB + 20 µL ampicillin + 20 µL kanamycin + 10 µL inoculum + 32 µL dexamethasone (1:1000) (inoculate ON).

We ran a gel for several PCRs: F-H-P-E1GF2-MP-E2GF-E3GF1-E3GF2-E4GF- Everything was succesful except E1GF2.

Then we digested the PCRs with 5 µL Buffer CutSmart + 15 µL PCR + 1 µL XbaI + 1 µL SpeI + 28 µL water (for each PCR)