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. Miniprep
    1. Throw the supernatant
    2. resuspend in 205 uL of resuspension solution in an Eppendorf
    3. Add 250 uL of lysis solution for 2 min then add 350 uL of neutralization solution and shake it hard
    4. 10 min of centrifugation at 14k rpm
    5. supernatant is pour in a column and centrifugated for 30 sec at 14k rpm in a column
    6. supernatant is discarded and 700uL of washing solution are added then the column is centrifugated at 14k rpm for 30s
    7. supernatant is discarded and 500uL of washing solution are added then the column is centrifugated at 14k rpm for 30s
    8. supernatant is discarded, the column is centrifugated at 14k rpm for 120s
    9. put the column in another tube and add 45uL of DNAse RNAse free water in the middle of the column
    10. wait 2 min
    11. centri for 2 min at 10k rpm
    12. discard the column
  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.
  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: from left to right dilutions
  • from left to right: dilutions 1/1 1/10 1/100
  • first line: without g418 second line: with g418












22/07
Received gBlocks vA-2, vA-3 and vA-4, which form the last parts of the polycistron, and the corresponding amplification oligos from IDT.
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 amplification, using the following protocol:

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

We made 2 PCR tubes for each gBlocks, with the following primers:
vA-2 + o15.056 + o15.057
vA-3 + o15.058 + o15.059
vA-4 + o15.060 + o15.061

Settings PCR:
35 cycles amplification, using the following parameters:

time (min) temperature (°C) function
0:30 98 melting
0:10 98 melting
0:30 50 annealing
1:00 72 extension
10:00 72 extension
forever 10 storage
After PCR, we migrated the PCR product on TAE 1% agarose gel (100V), to check if the amplification product had the expected size.
From left to right:
100bp+ ladder, vA-2 (two wells), vA-3 (two wells), vA-4 (two wells) amplified at 52°C

We then made a PCR purification using QIAGEN kit:

    PCR purification protocol

  • Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down
  • Transfer in a centrifugation column
  • Centrifuge 2 min at 14000 rpm
  • Discard flow-through
  • Add 700μL of washing solution
  • Centrifuge 30 sec at 14000 rpm
  • Discard flow-through
  • Add 500μL of washing solution
  • Centrifuge 30 sec at 14000 rpm
  • Discard flow-through
  • Centrifuge 30 sec at 14000 rpm
  • Discard flow-through
  • Put the column in a sterile 1.5 mL Eppendorf tube
  • Add 45μL of DNAse/RNAse-free water on the membrane
  • Wait 2 minutes
  • Centrifuge 2 min at 10000 rpm
  • Discard column, DNA is saved in water

DNA concentration measured with Nanodrop:
Part Name PCR (ng/μL)
vA-2 (tube 1) 126
vA-2 (tube 2) 97
vA-3 (tube 1) 129
vA-3 (tube 2) 76
vA-4 (tube 1) 202
vA-4 (tube 2) 108

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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 (NEB turbo) that was kept at -80°C, with our Gibson product, and also with the HO-Poly-KanMX4-HO vector alone to make a control. The electroporation was realised following the electroporation protocol described in the protocol page of the wiki. The bacteria recovered for 3 hours in SOC media after the electroporation. 100uL of the bacterial cultures were then plated on LB with or without Amp with different concentrations: 10^-1 10^-2 and 10^-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 very well (a lawn of bacteria on the plates with the 10^-1 and 10^-2 dilutions and more than 100 CFU on the 10^-3 plate.

    Interpretation
    What most likely happen is that the Gibson assembly failed.
    Or maybe the Gibson Assembly worked, but we shouldn’t have kept the product overnight before transforming E. Coli with it. Now the plan is to make all the PCR again

    plan of the PCR

    • gBlock 1.1 with primers o15.141 and o15.144
    • gBlock 1.2 with primers o15.119 and o15.120
    • gBlock 2 with primers o15.127 and o15.128
    • gBlock 3 with primers o15.129 and o15.130
    • gBlock 4 with primers o15.131 and o15.132
    • HO-Poly-KanMX4-HO with primers o15.135 and o15.143

    results of the PCR

    • migration on a gel
    • 5uL of each sample was mixed with 1uL of loading dye then run on a gel with TAE for 20 min

      • Results

    • 1uL of each sample was nanodroped and the concentrations are shown below

    Case of the gBlock 3

    because it does not amplify well and is showing signs of unspecific binding we are performing a gel purification on this gBlock.

    PCR and gel purification


    6 tubes of 100 uL were made following the PCR protocol described in the protocol part of the wiki (with elongation time=1:30 min, enzyme=phusion, 35 cycles, 57°C annealing temperature)
    PCR product is then put on a gel to migrate and extracted using the gel purification protocol that is presented in the protocol page of the wiki.
    all the product is then purified using the PCR purification protocol described in the wiki.
    the concentration of the DNA is then checked using the nanodrop.