Team:Paris Bettencourt/Notebook/VitaminA

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

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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 the following result (on the right):
There are clear bands at 1.5 kb, so 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

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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
______

Aug. 5th
PCR gBlocks vA-1.1 and vA-1.2 with tails and PCR HO-Poly-KanMX4-HO plasmid.
Aug. 6th
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. We calculated the required amount of each insert to put on the Gibson mix, and made a separate tube called “gBlock mix” containing the required concentration of each gBlock in 100 uL.
    Titre du tableau
    Component (size) [PCR product] Quantity required for Gibson Volume put in “gBlock mix”
    vA-1.1 (701 bp) 84 ng/uL 13 ng 15.5 uL
    vA-1.2 (654 bp) 120 ng/uL 13 ng 10.8 uL
    vA-2 (1560 bp) 126 ng/uL 25 ng 19.8 uL
    vA-3 (1530 bp) 129 ng/uL 25 ng 19.4 uL
    vA-4 (1584 bp) 108 ng/uL 25 ng 23.1 uL
    plasmid HO-Poly-KanMX4-HO (6 kb) 103 ng/uL 100 ng -

    The “gBlock mix” contained 88,6 uL with all the gBlocks, and we added 11.4 uL of water to reach 100 uL.
    The final Gibson mix contained:
    • 15 uL Gibson master mix
    • 1 uL HO plasmid at 100 ng/uL
    • 1 uL “gBlock mix”
    • 3 uL water
    Total = 20 uL.
    The solution was put at 50°C for 60 minutes, then stored at 4°C overnight.
    Aug. 7th
    Transformation
    We transformed by electroporation an E. Coli (from NEBT. Name = ???) that was kept at -80°C, with our Gibson product, and also with the HO-Poly-KanMX4-HO vector alone to make a control. Protocol of electroporation: JB à toi de remplir ça :3

    Aug. 8th

    Results
    We didn’t have any colonies after the overnight culture of the cells transformed with the Gibson product. The E. Coli transformed with the HO-Poly-KanMX4-HO vector alone did grow though.

    Interpretation
    It’s possible that the Gibson Assembly didn’t work, because… ???
    Or maybe the Gibson Assembly worked, but we shouldn’t have kept the product overnight before transforming E. Coli with it.