Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminA"

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Jul. 14th

Goal

Extract the integrative plasmid HO-Poly-KanMX4-HO from the E.Coli provided by AddGene (Accession number #51662).

Procedure

1. Liquid culture overnight in LB + Ampicillin.
2. Made a glycerol stock and stored it in the -20 freezer (g15.35)
3. Centrifuge the tube for 1 minute with 11000 rpm.
4. Follow the standard protocol of Thermo Scientific miniprep kit (is it? I followed Ihab's protocol here: https://docs.google.com/document/d/1QHBHcN8RY0c-oEY8X5kAqPrKYODTBm7K8eU_lY_yw-o/edit, we should compare them)
5. Measure concentration with Nanodrop

Results

Final DNA concentrations of the 4 tubes of miniprep, measured with Nanodrop:

• tube HO pl. 1 = 431.1 ng/uL
• tube HO pl. 2 = 313.6 ng/uL
• tube HO pl. 3 = 366.4 ng/uL
• tube HO pl. 4 = 261.7 ng/uL

______


Jul. 15th

Goal

Test chromosomal integration in WT yeast SK1 with the integrative plasmid HO-Poly-KanMX4-HO.

Procedure

We followed the method described in "High-efficiency Yeast transformation using liAc/SS carrier DNA/PEG method" (Gietz 2007).

1. Inoculation of a single colony of the SK1 yeast strain in liquid YPD overnight on a rotatory shaker at 130 r.p.m and 30°C.
2. After 16 hours the titer of the cell culture was determined. The OD 600 nm of a 1/100 dilution (10 uL in 1 mL) was measured and the cell concentration determined using the formula (1*10^6 cells.mL^-1 will give an OD600nm of 0.1).
3. 2.5*10^8 cells were added to 50 mL of pre-warmed YPD and incubated for 4.5 hours at 30°C and 130 rpm.
4. 1.0 mL of salmon sperm carrier DNA was denaturated in a heat block at 99°C during 5 min, and chilled rapidly in ice.
5. The cells where harvested by centrifugation at 3000g for 5 min and resuspended in 25 mL of water and centrifuged again 5 min at 3000g. The washing process was repeated again, then the cells were resuspended in 1.0 mL of sterile water.
6. The suspension was transfered into a 1.5 mL eppendorf tube and centrifuged for 30s at 13,000g and the suppernatent discarded
7. The cells were resuspended in 0.5 mL of sterile water. 100uL of the solution are pipette in a 1.5mL microcentrifuge then the transformation mix has been added(240uL of PEG 3350,36uL of liAc 1.0 M, 50 uL of the single stranded DNA carrier(2.0 mg.mL^-1, 35 uL of DNA plus sterile water up to a total of 360 uL.u woot m8 ?
8. tubes were placed at 42°C for 40 min.
9. tubes were centrifuged at 13 000g for 30s in a microcentrifuge and the supernanant removed with a micropipettor. Pellet was resuspended in YPD and incubate for 3 hours at 30°C to ensure good expression of the antibiotic resistance.
10. 2,20 and 200 uL of the cell suspension were plated on YPD agar + G418 and spread with glass beads.
11. plates were put to grow at 30°C for 3 days

Results

We had many transformants, with the resistance marker:
[add photos]
we must not forget to mention the controls.











Jul. 22nd

Goal

Amplify gBlocks vA-2, vA-3, and vA-4, which form the last parts of the polycistron.

Procedure

Resuspend gBlocks and primers:

• Resuspended gBlocks vA-2, vA-3 and vA-4 in 100 uL water, to reach a final concentration of 10 ng/uL.
• Made aliquots of these gBlocks at concentration 1 ng/uL.
• Resuspended primers o15.056, o15.076, o15.058, o15.059, o15.060, o15.061 in water to reach a final concentration of 100 uM for each.
• Made aliquots of each primer at concentration 10 uM.

PCR: Made 2 tubes of PCR for each gBlocks, containing:

• 1 uL of gBlock (1 ng)
• 2 uL forward primer
• 2 uL reverse primer
• 50 uL Master Mix 2X
• 45 uL water

Settings PCR:

• 30s at 98°C
• 35 times:
  • 10s at 98°C
  • 30s at 50°C
  • 1m at 72°C
• 10m at 72°C
• the tubes were then kept at 10°C

Results

After gel migration, we got this: [image]
The PCR worked.

We then made a PCR purification (should I describe full protocol?) and measured the final concentration of gBlocks with a Nanodrop:

• Final concentration gBlock vA-2 tube a = 126.1 ng/uL
• Final concentration gBlock vA-2 tube b = 96.6 ng/uL
• Final concentration gBlock vA-3 tube a = 128.7 ng/uL
• Final concentration gBlock vA-3 tube b = 75.8 ng/uL
• Final concentration gBlock vA-4 tube a = 201.8 ng/uL
• Final concentration gBlock vA-4 tube b = 108.3 ng/uL

______


Jul. 28th

Goal

Retrieve TDH3 promoter from Biobrick BBa_K530008.

Procedure

PCR of TDH3:

• 1 uL of gBlock (1 ng)
• 2 uL forward primer o15.123
• 2 uL reverse primer o15.124
• 50 uL Master Mix 2X
• 45 uL water

Settings PCR:

• 30s at 95°C
• 35 times:
  • 30s at 95°C
  • 30s at 55°C
  • 1m at 72°C
• 10m at 72°C
• the tubes were then kept at 10°C

Results

Nanodrop : 57,9 ng/uL
______

Gibson assembly

The goal is to assemble all the parts together to get the plasmid with the 3 genes that are needed to produce beta carotene in Saccharomyces cerevisiae.
Gibson was performed on :
• the PCR product of HO-Poly-KanMX4-HO, a plasmid from addgene
• PCR product of the gblock 1.1
• PCR product of the gblock 1.2
• PCR product of the gblock 2
• PCR product of the gblock 3
• PCR product of the gblock 4

5X ISO Buffer was prepared with the following recipe:
3 ml 1M Tris-HCl pH 7.5
+ 150 μl 2 M MgCl2
+ 240 μl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)
+ 300 μl 1 M DTT
+ 1.5 g PEG-8000
+ 300 μl 100 mM NAD
dH20 to 6 ml

Prepare 1.2 ml of Gibson assembly master mix as follows:
320 μl 5X ISO Buffer
+ 0.64 μl 10 U/μl T5 exonuclease*
+ 20 μl 2 U/μl Phusion polymerase
+ 160 μl 40 U/μl Taq ligase
+ _ dH20 to 1.2 ml
We took 15 ul of the mix for our Gibson, and stored the rest at -20 C in 15 μl aliquots.

Titre du tableau

Component (size)	[PCR product]	Quantity required
vA-1.1 (701 bp)	84 ng/uL	13 ng
vA-1.2 (654 bp)	120 ng/uL	13 ng
vA-2 (1560 bp)	126 ng/uL	25 ng
vA-3 (1530 bp)	129 ng/uL	25 ng
vA-4 (1584 bp)	108 ng/uL	25 ng
plasmid HO-Poly-KanMX4-HO (6 kb)	103 ng/uL	100 ng