Team:Paris Bettencourt/Notebook/VitaminA

July 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 out the supernatant
    2. resuspend in 250 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 lightly.
    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-Poly-KanMX4-HO pl. 1 = 431.1 ng/uL
  • tube HO-Poly-KanMX4-HO pl. 2 = 313.6 ng/uL
  • tube HO-Poly-KanMX4-HO pl. 3 = 366.4 ng/uL
  • tube HO-Poly-KanMX4-HO pl. 4 = 261.7 ng/uL


July 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 supernatant 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 1/1 1/10 1/100
  • first line: without g418
  • second line: with g418


July 22nd


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 product] (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


July 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

August 3rd

We made the following PCR:
vA-1.1 + o15.142 + o15.141
vA-1.2 + o15.119 + o15.122
HO-Poly-KanMX4-HO plasmid + o15.135 + o15.143
The elongation time during the pCR was 1 minute for the two gBlocks, and 4 minutes for the plasmid.

August 4th

Results:
We migrated all the PCR products of our gBlocks on a gel:

The bands for gBlocks vA-1.1, 1.2, 2 and 4 are at the expected position. The band for vA-1.1 is not very bright but still visible. There seems to be some unspecific binding for gBlock vA-3 as we can see two bands, one at the expected size and one smaller. We still decided to go on and try our Gibson Assembly, assuming the smaller DNA sequence wouldn't be that much of a problem as long as the right PCR product of gBlock vA-3 was present too.

DNA concentration was measured with a Nanodrop:
Part Name [PCR product] (ng/μL)
vA-1.1 84
vA-1.2 22
HO-Poly-KanMX4-HO 25


August 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 vA-1.1
  • PCR product of the gblock vA-1.2
  • PCR product of the gblock vA-2
  • PCR product of the gblock vA-3
  • PCR product of the gblock vA-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.
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-Poly-KanMX4-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.

August 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

August 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, which could be due to the unspecific binding during the PCR of gBlock vA-3, or to secondary structures at the extremities of our gBlocks which could have made the binding difficult.
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, to make fresh electro-competent cells, and to Gibson and transform on the same day.

August 10th

Plan of the PCR

  • gBlock vA-1.1 with primers o15.141 and o15.144
  • gBlock vA-1.2 with primers o15.119 and o15.120
  • gBlock vA-2 with primers o15.127 and o15.128
  • gBlock vA-3 with primers o15.129 and o15.130
  • gBlock vA-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

    • We can observe the expected bands for most of the gBlocks, except for gBlock vA-3 for which there is no clear band, and gBlock vA-1.1 which has a additional, unexpected band at a lower size, probably due to an unspecific binding of an oligo.
  • 1uL of each sample was nanodroped and the concentrations are shown below:
    • Part Name [PCR product] (ng/μL)
    vA-1.1 204
    vA-1.2 95
    vA-2 77
    vA-3 77
    vA-4 134
    HO-Poly-KanMX4-HO 61

Case of the gBlock vA-3

Since the gBlock vA-3 didn't amplify well on the last PCR, we tried to amplify it again, and this time observed 3 bands, showing unspecific binding of the oligos. We performed a gel purification on this gBlock to take only the DNA in the band at the right size.
On a une photo avec triple bande du gBlock 3 ? Sur la photo au-dessus y a plutôt 0 bande...

PCR and gel purification

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

August 13th


We put some E. Coli NEB-Turbo to grow overnight in 2 mL LB, at 37°C with shaking.

August 14th


We made our E. Coli NEB-Turbo electrocompetent with the following protocol:
    Preparation of electrocompetent cells protocol

  • The night before the transformation, start an overnight culture of cells.
    • 5 ml LB.
  • The day of the transformation, dilute the cells 100X.
    • 100 ml LB.
    • Grow at 37°C for about 2 hours.
  • Harvest the cells.
    • When the cells reach an OD600 of between 0.6 and 0.8.
    • Split the culture into 2x 50 ml falcon tubes, on ice.
    • Centrifuge at 4 °C for 10 min at 4000 rpm.
  • Wash and combine the cells.
    • Remove the supernatant.
    • Resuspend the cells in 2x 25 ml of ice cold water.
    • Combine the volumes in a single 50 ml falcon tube.
  • Wash the cells 2 more times.
    • Centrifuge at 4 °C for 10 min at 4000 rpm.
    • Resuspend in 50 ml of ice cold water.
    • Repeat.
  • Wash and concentrate the cells for electroporation.
    • Centrifuge at 4 °C for 10 min at 4000 rpm.
    • Resuspend in 1-2 ml of ice cold water.
    • We will use 200 ul of washed cells per transformation.

August 24th

PCR gBlock 3 with primers o15.129 and o15.130 3 tubes with concentrations of gBlock of 1ng 1ng and 10ng

August 25th

After running on a gel the DNA is Gel Purified. The Nanodrop measurement then revealed that everything has been lost during the gel purification. Another PCR is launched with all the gBlocks and the right tails.

plan of the PCR

  • gBlock vA-1.1 with primers o15.141 and o15.142
  • gBlock vA-1.2 with primers o15.119 and o15.120
  • gBlock vA-2 with primers o15.127 and o15.128
  • gBlock vA-3 with primers o15.129 and o15.130
  • gBlock vA-4 with primers o15.131 and o15.132
  • HO-Poly-KanMX4-HO with primers o15.135 and o15.143


We also tried a Gibson Assembly again with the amplified gBlocks vA-1.1, 1.2, 2, 4 and the HO-Poly-KanMX4-HO vector ... which failed miserably.

on the right: 1kb ladder
on the left: Gibson product
no band at 1500 bp is visible (the exposition time is 5s)


August 26th

result of the PCR of the gBlocks (from left to right) vA-1.1, 1.2, 2, 3, 4, HO-Poly-KanMX4-HO and the last column is the ladder 1kb

The PCR for the gBlock 1.2, 2 and 4 worked well.
We are doing again the PCR for the gBlock 1.1, 3 and HO
The PCR with the gBlock 3 is done with 10ng of DNA
from left to right: gBlock 1.1, 3 (1ng), 3 (10ng), HO, 1kb ladder
The results are showing traces of unspecific binding for 1.1 and 3. The HO-Poly-KanMX4-HO band is alsmost invisible, predicting a low DNA concentration.


August 27th

Miniprep of the overnight cultures of the small and big colonies obtained after transformation of SOPHIIIIEEE ICIIII JE MET QUOIIII ? *****
Following the miniprep an analytical digest is performed with Pst1 to linearise the plasmid and then by Nde1 which is supposed to cut the plasmid in 2.
During the digestion the miniprep DNA concentration is recorded with a Nanodrop
colony which was grown for the miniprep S1 S2 S3 S4 B1 B2 B3 B4
Nanodrop concentration
(ng.ul-1)		 84 176 182 183 128 216 170 235

Then the results are tested with a gel migration.

GEL 1
from left to right 1kb ladder, small colony 1,2,3,4, big colony 1,2,3,4, small colony digested by Pst1 1,2,3,4, big colony digested by PST1 1,2,3,4


GEL 2
from left to right 1kb ladder, small colony digested by Nde1 1,2,3,4, big colony digested by Nde1 1,2,3,4



August 28th

PCR on the minipreped plasmid.


PCR with the oligos o.15.193 and o.15.194 and 10 ng of plasmid DNA.
The PCR product are then revealed with a gel electrophoresis.


from left to right: 1kb ladder thermoscientific, PCR of the plasmid on 4 different colonies(s2, b2, s4, b4)
The brightest band (at 2kb)is probably the band that we were expecting after PCR (the DNA piece that i supposed is amplified is composed of the promoter, the terminator and the insert).