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Let's PLAy project - Bioproduction of PLA

Note book



Complete version of our note book (.pdf)


Mon Tue Wed Thu Fri Sat Sun
July
Week 1 4 5 6 7 8 9 10
Week 2 11 12 13 14 15 16 17
Week 3 18 19 20 21 22 23 24
Week 4 25 26 27 28 29 30 31
August
Week 5 1 2 3 4 5 6 7
Week 6 8 9 10 11 12 13 14
Week 7 15 16 17 18 19 20 21
Week 8 22 23 24 25 26 27 28
Week 9 29 30 31
September
Week 9 1 2 3 4
Week 10 5 6 7 8 9 10 11
Week 11 12 13 14 15 16 17 18
Rest of the lab book (.pdf)

July 2016


Week 1


4th of July

Bottles of LB and LB-Agar (200 mL and 400 mL)
  • LB-Agar: 14 g for 400 mL of water
  • LB: 8 g for 400 mL of water
  • Autoclave
Resistance test of P. putida

2 plates of LB-Agar with either Kanamycin (50 µg/mL) or Spectinomycin (50 µg/mL).

Preparation of HEPES solution 0.5 M (for electroporation)
  • 11 g of HEPES (solid) in 64 mL of water
  • NaOH (0.1 M) for pH7
Refresh P. putida (the Petri dish got old)

E. coli C118 with plasmids pSEVA2311 (KanR) and pSEVA224 (KanR) were separately spread in LB.

  • cells with pSEVA2311 were observed
  • cells with pSEVA224 were not observed!!
  • Jonathan asked for another sample

5th of July

  • LB culture of E. coli C118
  • HEPES filtration
  • Check the inventory of the fridge
  • Plate of P. putida at 30 °C and 37 °C → We didn’t do this step
  • Liquide culture (5 mL of preculture) of P. putida to have competent cells

6th of July

Preculture for transformation (5 mL) of E. coli DH5 α

7th of July

Miniprep of E. coli C118 with pSEVA2311
  • Protocol “Midiprep” with 50 mL of culture
  • C = 80 ng/µL in 200 µL of elution buffer
  • OD260 = 1.616
  • OD280 = 0.8
Transformation of Pseudomonas with pSEVA2311
  • 40 mL of culture (competent cell):
  • When OD600nm = 0.6
  • 20 mL for Heat Shock
  • 20 mL for Electroporation

After electroporation and heat-shock spread on Amp-Plates

8th of July

The transformation of 07/07/2016 didn’t work because the plasmid given by Robert didn’t match with Pseudomonas→ Second transformation (Heat shock and electroporation)

Preparation of plates (LB + Antibiotics)
  • 10 plates LB-Agar, Kanamycin (1 µL/mL)
  • 10 plates LB-Agar, Chloramphenicol (1 µL/mL)
  • 10 plates LB-Agar, Ampicillin (1 µL/mL)
  • 10 plates LB-Agar, without antibiotic

Week 2


12th of July

Preparation of M9-media (10X, V= 600 mL) with different kind of C-sources
  • Glucose → DONE
  • Glycerol → DONE
  • Fructose → NO YET
  • Benzoate → NO YET
  • Lactic acid → NO YET
  • Methane → NO YET

Compounds Cf Masses (g) or volumes (ml)
CaCl2.H2O 1 mM 0.0882 g
MgSO4 20 mM 1.44 g
FeSO4.H2O 0.1 mM 0.01668 g
M9 (salt) 10 X 67.68 g
Glycerol (100 %) * 8 % v/v 60.48 g
Casamino acid 2 % w/v 12 g
Thiamine 10 mg/mL ** 10 µg/mL (0.6 g)
Uracil 200 µg/mL 0.12 g
Leucine 300 µg/mL 0.18 g
NaOH pH 6.6 -

Table 1. *Glucose : 59.22 g;
** Thiamine has not been added but it has to be before the preculture of P. putida.

→ problem : precipitation
13th of July

Preparation of the deletion process – Transformation of E. coli Mach1 competent cells (CaCl2 treatment) by heat shock
  • Plasmids pKD46, pCP20 and pKD4
  • 3 tubes in which 25 µL of cells were mixed with 1.5 µL of each plasmid (pKD4, pCP20 or pKD46)
  • Incubation on ice for 20 min
  • Heat at 42 °C (thermobloc) 1 min
  • 120 µL of LB added to each tube
  • Incubation: 30 min at 37 °C for the plasmid pKD4, 30 °C for the plasmids pCP20 and pKD46
  • Spread on plates (Ampicilline 100 μg/mL)
  • Incubation overnight at 37 °C for pKD4, 30 °C for pCP20 and pKD46
Preparation for the deletion process – Preparation of E. coli DH5 α competent cells
  • 5 mL of preculture of DH5 α were prepared
  • Incubation overnight at 37 °C
14th of July

  • pKD46 and pCP20 transformation properly worked
  • pKD4 didn’t succeed
  • Preparation of 5 mL culture of pKD46 and pCP20 to purify plasmid
  • pKD4 since it is not needed for transformation will be used w/o amplification
  • DH5 α competent cells were prepared and stored
15th of July

Midiprep of pCP20 and pKD46
  • pCP20: 65 ng/µL
  • pKD46: 50 ng/µL
Transformation of P. putida KT2440 with pKD46 plasmid to start deletion process
  • Incubation overnight at 30 °C (Amp Plates)
  • Spread KT2440 WT and check growth at 37 °C
16th of July

Transformation results
  • cells grew on Ampicillin at 30 °C
  • Pick 4 clones from previous plate and spread them in a new plate (Amp with proper concentration)
  • Incubation at 30 °C
Pseudomonas putida : Working concentration of Antibiotics
  • Kan (50 µg/mL) → 1.7 µL of stock solution/mL
  • Amp (500 µg/mL) → 5 µL of stock solution/mL
  • Spec (50 µg/mL) →1 µL of stock solution/mL

KT2440 grew well at 37 °C

17th of July

Week 3


18th of July

Electroporation of P. putida : pKD46
  • Electro-competent cells have been prepared
  • 1.5 µL of DNA were added to ~ 50 µL of electro-competent cells (all the tube)
  • 1 mL of LB was transfer in a 1.5 mL tube
  • 500 µL of the LB were prepare in the pipette-tip and put rapidly in the electro-cuve after electroporation (at 1800V)
  • All the cells+LB of the cuve were transferred to the 1.5 mL tube containing LB
  • Incubation + Shaking 1 h at 30 °C
  • Centrifugation 3 min 90 rpm
  • Cells were plated in LB Agar Ampicillin (100 μg/μL) and incubated at 30 °C
Spread C118 (pSEVA224) on plates with Kanamycin
19th of July

The transformation (Electroporation of 18/08) didn’t work → maybe the replicon doesn’t match with P. putida

Colony PCR: to check the transformation of P. putida with p2311
  • Tick one colony and with the same tip, spread a little area on LB plate and dissolve the rest in 10 µL ddH2O
  • Gently vortex and briefly centrifuge DreamTaq Green PCR Master Mix (2X) after thawing
  • Place a thin-walled PCR tube on ice and add the following components for each 50 µL reaction

DreamTaq Green PCR Master Mix (2X) 1X 25 µL
Forward primer 0.5 µM 0.25 µL
Reverse primer 0.5 µM 0.25 µL
Colony water ---- 2 µL
Water, nuclease-free ---- 22.5 uL
  • Gently vortex the samples and spin down
  • Perform PCR using the recommended thermal cycling conditions outlined below

Step Temperature °C Time Number of cycles
Initial denaturation 95 °C 5 min 1
Denaturation 95 °C 30 s 30
Annealing 55(Tm)-5 °C 30
Extension 72 °C 1 min/kb
Final extension 72 °C 10 min 1
Pause 4 °C “00” ---
  • Load 5-15 µL of PCR mixture directly on a gel
Electrophoresis to check the transformation
  • 1 % of agarose
  • Duplicates have been done

See complete version of the notebook for details on the conditions.

gel 1
PCR of pKD4

Mix for 100 µL


Mix 1
Reaction buffer 20 µL
dNTP mix 2 µL
Primers (reverse and forward) 0.5 µL
Template (pKD4) 1 µL
Q5 polymerase 1 µL
H2O Qs 100 µL (75.5 µL)

Cycles
Temperature Time Number of cycles
98 °C 30” 1
90 °C 30”
60 °C 30” 32
72 °C 1’
72 °C 5’ 1
4 °C “00” ---

Culture of E. coli C118 pSEVA for midi prep’
20th of July

Midiprep’ of pSEVA224 (from E. coli)

C= 59.9 ng/µL in 200 µL of elution buffer

DO260/DO280 = 1.97

Alternative to delete gene

Transformation of Pseudomonas by a PCR product : Kanamycin resistance cassette + homologous region 5’ and 3’ using Heat Shock and Electroporation.

  • See protocols for both transformations
  • We used 10 µL of DNA
Preparation of 7 plates for Pseudomonas (1.7 µL/mL of kanamycin stock solution)
Liquid culture of E. coli pSEVA224 (2 mL)
  • 2 mL of LB
  • 3.4 µL of Kan - It was a mistake: we should have put 2 µL of Kan (since we used E. coli)
Preculture to amplify the vector given by Cyril (BBa.R0010 pLac)
  • E. coli G3A
  • 50 mL of LB
  • 50 µL of Amp
21th of July

!!! The fridge and freezer went off !!!
The transformation of the PCR product (for the deletion process) didn’t worked
  • We will focus on the construction of the plasmid
Store the culture

Storage in glycerol = 1 vol of glycerol + 1 vol of the culture and at -80 °C

Midiprep of the vector R0010
  • 325 ng/µL
  • DO260/DO280 = 1.8
Preparation of Ampicillin solution stock 50 mg/mL in H2O (Helix) aliquoted in 5 tubes of 2 mL
  • In sterile condition
  • 0.5 g of Amp + 10 mL of H2O
  • Filtration (0.2µm)
  • Storage away from light
Digestion of
  • the vector (with Ba.R0010 pLac from Cyril) by EcoRI (E) and SpeI (S)
  • the vector (with Ba.R0010 pLac from Cyril) by EcoRI and PstI (P)
  • the phaC sequence by EcoRI and SpeI
  • the Propionyl-CoA transferase sequence by EcoRI and PstI

  • By the way, we tried to remove the promoter (BBa.R0010 pLac) from the vector
  • 100 µL of Water were added to the phaC sequence and the Propionyl-CoA transferase sequence
Vector (E/S) phaC (E/S) Vector (E/P) propCoA(E/P)
DNA 2 µL 10 µL 2 µL 10 µL
Buffer Cutsmart 10X: 3 µL Cutsmart 10X: 3 µL 2.1: 3 µL 2.1: 3 µL
Enzymes EcoRI: 1 µL EcoRI: 1 µL EcoRI: 1 µL EcoRI: 1 µL
SpeI: 1 µL SpeI: 1 µL PstI: 1.5 µL PstI: 1.5 µL
H2O 23 µL 15 µL 22.5 µL 14.5 µL

1 h at 37 °C

Electrophoresis
Wells: Marker Control vector with pLac Vector E/S (30 µL DNA + 6 µL loading buffer) Vector E/S (30 µL DNA + 6 µL loading buffer)
(8 µL) (5 µL DNA + 1 µL loading buffer)
Expected results __ 1 band (2270 bp) 2 bands (2070 bp + 200 bp) 2 bands (2070 bp + 200 bp)

  • 1 % agarose
  • Vector (with pLac): 2270 bp
  • Promoter pLac ~ 200 bp
Results

* : circular plasmid (3 forms: compacted, intermediate and loose forms)

  • -We get rid of the promoter and we kept the empty vectors
Purification of the DNA sequences phaC and prop-CoA transf.
  • Zymoclean Gel DNA Recovery Kit
  • Elution with 6 µL of water
  • Wait 1-2 min before the last spin
  • The check at the spectrophotometer nanodrop showed a pic around 240 nm
  • Maybe: problem with the wash buffer reminded in the column
  • Inversion of the 2 columns before the second spin
22th of July

Ligations
Empty vector (R0010) 10 µL
Insert phaC 20 µL
T4 buffer 10X 4 µL
T4 ligase 2 µL
H2O 5 µL

Empty vector (R0010) 10 µL
Insert propCoA transf. 20 µL
T4 buffer 10X 4 µL
T4 ligase 2 µL
H2O 5µL

  • Incubation 1 h at RT
Transformation of E. coli DH5 α, Top 10 and BL21 by Heat Shock
  • Same protocol
  • We didn’t recover enough DNA, insert from previous midiprep, purification
  • These strains were spread on labeling plates (see table below)
  • Incubation overnight at 37 °C
22/07/16 E. coli phaC DH5 α 22/07/16 E. coli phaC Top10 22/07/16 E. coli phaC BL21
22/07/16 E. coli propCoA DH5 α 22/07/16 E. coli propCoA Top10 22/07/16 E. coli propCoA BL21
23th of July

  • “Transformed” clones were observed
  • Colony PCR

Week 4


25th of July

Media
  • 5 bottle of LB
  • 3 x 400 mL
  • 2 x 200 mL
  • 5 bottle of LB-Agar
  • 3 x 400 mL
  • 2 x 200 mL
Previous transformations (22/07) worked
Electrophoresis (PCR colony 23/07)
  • Gel 1 %
  • propCoA (in Cyril’s vector)

Gel 3

  • phaC (in Cyril’s vector)

Gel 4

Precultures
  • 2 mL of LB
  • Amp (1 µL stock solution/mL)
  • A good colony
  • phaC : 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20
  • propCoA :1, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
  • Incubation overnight at 37 °C
26th of July

Miniprep’ kit Sigma-Aldrich : extraction of the plasmids from the precultures of the 26th of july
  • For the propCoA transferase : problem with the 8th tube we lost a lot of bacteria*
Colonies (with the phaC ) Concentrations of DNA (ng/µL) Colonies (with the phaC ) Concentrations of DNA (ng/µL)
1 282.3 11 160.9
2 170.4 12 173.0
4 174.6 13 170
5 176.5 14 185.3
6 188.9 15 162
7 167.8 16 189.5
8 185.9 17 193.6
9 191.4 19 183
10 173.4 20 163

Colonies (with the propCoA ) Concentrations of DNA (ng/µL) Colonies (with the propCoA ) Concentrations of DNA (ng/µL)
1 150.5 13 169.4
3 143.4 14 282.8
4 156 15 192.3
5 151.8 16 226
6 234.2 17 203.3
8 29.4* 18 175.1
10 194.5 19 256.5
11 167.2 20 181.7
12 162.8 - -

M9 medium 2 x 50 mL of 2.5 X
  • Without C-sources, thiamine
  • Problem: precipitation (the same than previous M9 media)
27th of July

Stock solutions
  • CaCl2 1 M: 27.745 g in 250 mL of milliQ
  • MgSO4 1 M: 30.1 g in 250 mL of milliQ
  • FeSO4 10 mM: 0.379 g in 250 mL of milliQ
Preculture of Pseudomonas putida KT2440 (2 mL)
28th of July

Aliquots for sequencing V= 20 µL
Medium M9 1X without Carbone source and filtred (2 µm) in 2 falcon of 50 mL
  • Casamino acid : 1.2 g
  • Leucine: 3 mg
  • Uracil: 2 mg
  • Thiamine 10 mg/mL: 10 µL
  • M9 salt 5X: 20 mL
  • CaCl2 1 M: 10 µL
  • MgSO4 1 M: 100 µL
  • FeSO4 10 mM: 1 µL
  • Water milliQ qs 100 mL
  • NaOH for pH between 6 and 7.4 → no need since the pH was already at 6.8

No precipitation

10 mL of Media with C-source
  • 0.10037 g of Glycerol (100 %) (0.16 put) in 10 mL of milliQ water
  • 0.0982 g of Glucose in 10 mL of milliQ water
  • 0.057 g of Benzoic acid in 10 mL of milliQ water
Preparation for plate (96 wells) and Growth rate with the CLARIO star machine
  • 26.7 µL of the overnight preculture of Pseudomonas in medium M9 (with either Benzoic acid, Glucose or Glycerol) qs 1 mL to have around OD= 0.005
  • Plate mode, slow kinetic, DO600nm, T= 30 °C, shaking (200 rpm) before cycle of 10 min
1 2 3 4
A - Benzoic acid M9 medium Glucose M9 medium Glycerol M9 medium
B Benzoic acid M9 medium BE-M9 with cells BE-M9 with cells BE-M9 with cells
C Glucose M9 medium GLU-M9 with cells GLU-M9 with cells GLU-M9 with cells
D Glycerol M9 medium GLY-M9 with cells GLY-M9 with cells GLY-M9 with cells

August 2016


Week 5


2nd of August

Resolubilisation of IDT gBlocks in 100 µL water (Cf= 10 ng/µL)

Digestion

Mix:

PhaC2 PCT1 LDH1 pSB1A3
GBlock gene 10 µL 10 µL 10 µL 2 µL (≈600ng)
Buffer NEB 3 µL (2.1) 3 µL (CutSmart) 3 µL (2.1) 3 µL (CutSmart or 2.1)*
Enzyme 1 1 µL (EcoRI) 1 µL (EcoRI) 1 µL (XbaI) 1 µL (EcoRI or XbaI)
Enzyme 2 1 µL (PstI) 1 µL (SpeI) 1 µL (PstI) 1 µL (PstI or SpeI)
H2O (qs 30 µL) 15 µL 15 µL 15 µL 23 µL
  • *CutSmart when we doesn’t digest by PstI and 2.1 when PstI is used
  • Incubation 1 h 37 °C
Agarose gel 1 %
2-August_agarose-gel
Digestion

Mix:

- PhaC2 PCT1 LDH1
GBlock gene 10 µL 10 µL 10 µL
Buffer NEB 3 µL (2.1) 3 µL (CutSmart) 3 µL (2.1)
Enzyme 1 1 µL (EcoRI) 1 µL (EcoRI) 1 µL (XbaI)**
Enzyme 2 1 µL (PstI) 1 µL (SpeI)* 1 µL (PstI)
H2O (qs 30 µL) 15 µL 15 µL 15 µL

* PCT1 has a PstI restriction site in its sequence so the digestion was done by SpeI
** LDH1 has a EcoRI restriction site site in its sequence so the digestion was done by XbaI

  • Incubation at 37 °C
DNA extraction from gel

DNA clean-up → kit Monarch PCR and DNA cleanup (#T1030G) NEB

→ DNA Gel extraction kit (#T1020G) NEB

  • Incubation at 55 °C to dissolve gel
  • 400 µL buffer for 100 mg of agarose gel
  • DNA elution in 40 µL water
Ligation
  • 10 µL Vector (pSB1A3 digested)
  • 20 µL Insert digested
  • 4 µL 10X T4 DNA ligase buffer
  • 2 µL T4 DNA ligase
  • 5 µL H2O
  • Incubation RT 1 h
Transformation
  • Thaw 25 µL of competent DH5α E. coli
  • Add 10 µL of ligated DNA
  • 20 min incubation in ice
  • 1 min 42 °C
  • Add 120 µL of LB medium
  • Incubation 40 min 37 °C
  • Spread on plates LBA (Ampicillin)
  • Incubation 37 °C overnight
3rd of August

Colony PCR

Mix (50 µL total volume reaction)

  • 25 µL DreamTaq PCR Mastermix 2X (ThermoScientific)
  • 5 µL 10X DreamTaq Green Buffer (ThermoScientific)
  • 0.25 µL Forward Primer (0.5 µM) iG001
  • 0.25 µL Reverse Primer (0.5 µM) iG002
  • 19.5 µL H2O filtrated (qs 50 µL)

Mix prepared for 15 reactions (plus one reaction prepared without Green buffer)


PCR program:

  • 95 °C 5 min
  • (95 °C 30 s, 50 °C 30 s, 72 °C 2 min) 30 times
  • 72 °C 10 min
  • 10 °C ∞

Nanodrop
  • PhaC2 3.5 ng/µL
  • PCT1 3.8 ng/µL
  • LDH1 8.7 ng/µL
  • pSB1A3 (digested by X+P) 3.3 ng/µL

Gel electrophoresis

The results were not conclusive

4th of August

Colony PCR

Mix (25 µL total volume reaction)

  • 12.5 µL DreamTaq PCR Mastermix 2X (ThermoScientific)
  • 2.5 µL 10X DreamTaq Green Buffer (ThermoScientific)
  • 0.25 µL Forward Primer (0.5 µM) iG001
  • 0.25 µL Reverse Primer (0.5 µM) iG002
  • 9.5 µL H2O filtrated (qs 25 µL)

Mix prepared for 7 reactions


PCR program:

  • 95 °C 5 min
  • (95 °C 30 s, 50 °C 30 s, 72 °C 2 min) 30 times
  • 72 °C 10 min
  • 10 °C ∞

The results were not conclusive

Week 6


8th of August

Ligation
  • 50 ng vector
  • 5 µL T4 DNA ligase Buffer 10X
  • 3 µL T4 DNA
  • H2O qs 50 µL
  • DNA

PhaC 2 11.7 µL Vector E/P 7.8 µL H2O 22.5 µL
(3.5 ng/µL) (6.4 ng/µL)
PCT 1 30.4 µL Vector E/S 4.1 µL H2O 7.5 µL
(3.8 ng/µL) (12.2 ng/µL)
LDH 1 13.9 µL Vector X/P 8.5 µL H2O 19.6 µL
(8.7 ng/µL) (5.9 ng/µL)

We put 27.5 µL of Pct1 DNA because there was not enough.

Transformation
  • Thaw DH5-α competent bacteria in the ice.
  • Using the protocol processing by thermal shock.
  • 25 µL of bacteria + Mix of ligation (2 µL each time)
  • Incubation 20 minutes in ice and then 1 min at 42 °C
  • Adding 120 μl LB and incubate at 37 ° for 1 hour
  • Spread on LB Ampicillin with the öse
9th of August

Colony PCR

Mix PCR with 25 µL of total volume reaction prepared for 12 reactions prepared as before (DreamTaq #K1071 Thermo Scientific).

PCR program:

  • 95 °C 5 min
  • (95 °C 30 s, 50 °C 30 s, 72 °C 1 min) 30 times
  • 72 °C 10 min
  • 10 °C ∞

No bands on the gel (maybe problems with the enzyme)

10th of August

Digestion

Mix:

PhaC2 PhaC3 PCT2 LDH1
GBlock gene 20 µL 20 µL 20 µL 20 µL
Buffer NEB 3 µL (2.1) 3 µL (2.1) 3 µL (CutSmart) 3 µL (2.1)
Enzyme 1 1 µL (EcoRI) 1 µL (EcoRI) 1 µL (EcoRI) 1 µL (XbaI)*
Enzyme 2 1 µL (PstI) 1 µL (PstI) 1 µL (PstI) 1 µL (PstI)
H2O (qs 30µL) 5 µL 5 µL 5 µL 5 µL

* LDH1 has a EcoRI restriction site site in its sequence so the digestion was done by XbaI

pSB1A3 pSB1A3
Vector DNA 2 µL (≈600 ng) 6 µL (≈2 µg)
Buffer NEB 3 µL (CutSmart or 2.1)* 9 µL (CutSmart or 2.1)*
Enzyme 1 1 µL (EcoRI or XbaI) 3 µL (EcoRI or XbaI)
Enzyme 2 1 µL (PstI or SpeI) 3 µL (PstI or SpeI)
H2O (qs 30 µL) 23 µL 69 µL
Divided in 3 reaction tubes

Incubation 1 h 37 °C

1 % agarose gel
  • 2 trash nucleotides after the PstI restriction site in the suffix were forgotten so the digestion risks to fail → the digestion will be done by SpeI!
  • PhaC genes doesn’t match the gene in our reference publications but it corresponds to the gene used by the YALE IGEM team in 2013
11th of August

Extraction of digested vectors (pSB1A3 X+P and pSB1A3 E+P) from agarose gel (kit Zymoclean gel DNA Recovery kit #D4001S)

Concentration of the samples
  • pSB1A3 digested by X+P => 3.9 ng/µL
  • pSB1A3 digested by E+P => 7.2 ng/µL

Digestion by SpeI

Mix:

  • 20 µL genes digested by E+P or X+P or vector digested by X+P
  • 3 µL buffer NEB CutSmart
  • 1 µL enzyme SpeI
  • 6 µL H20 (qs 30 µL)

Or mix:

  • 20 µL genes digested by E+P or X+P or vector digested by X+P
  • 3 µL buffer NEB CutSmart
  • 1 µL enzyme SpeI
  • 6 µL H20 (qs 30 µL)

DNA purification
  • DNA clean & concentrator-5 (#D4003S Zymo Research)
  • For the vector: 2 volumes of binding buffer add to DNA (ratio 2:1)
  • For the genes: 5 volumes of binding buffer add to DNA (ratio 5:1)
  • Elution with 20 µL of water and incubation 2 min before centrifugation for DNA elution

Ligation

Mix:

PhaC2 PhaC3 PCT2
Vector pSB1A3 4.5 µL 4.5 µL 4.5 µL
Gene 14 µL 11 µL 10 µL
T4 DNA ligase Buffer 3 µL 3 µL 3 µL
T4 DNA ligase 1 µL 1 µL 1 µL
H2O (qs 30 µL) 7.5 µL 10.5 µL 11.5 µL

  • Incubation 1 h RT
  • Sample frozen at -20 °C until transformation
12th of August

Transformation
  • Thaw DH5-α competent bacteria in the ice.
  • Using the protocol processing by heat shock.
  • 25 µL of bacteria + 5 µL ligation mix
  • Incubation 20 minutes in ice
  • 1 min at 42 ° C
  • 3 min in ice
  • Adding 120 μl of LB
  • Incubation at 37 °C for around 50 min
  • Spread on LB Ampicillin plates
  • Incubation overnight 37 °C
13th of August

Results transformation

We got clones from the transformation. We took 3 clones from Pct(v2), 2 from phaC(v2) and 2 from phaC(v3) and performed PCR colony.

PCR colonies

20 µL mix per sample with:

  • 10 µL Green Taq Master Mix 2X (#K1081 Thermo Scientific)
  • 0.25 µL Forward primer iG001 (10 µM)
  • 0.25 µL Forward primer iG002 (10 µM)
  • 9.5 µL H2O (qs 20 µL)

Mix for 9 reactions + clones

PCR program:

  • 95 °C 5 min
  • (95 °C 30 s, 50 °C 30 s, 72 °C 2 min) 30 times
  • 72 °C 10 min
  • 10 °C ∞

1 % agarose electrophoresis (90 V, 111 mA, 40 min)

=> Result of PCR Colony: 1 clone of each was good

Precultures

3 mL LB medium + positive clones for each gene (PhaC2, PhaC3, PCT2)

Incubation 37 °C with shaking (around 16 h incubation)

Transformation (E. coli DH5-α with pSB1A3 ligated with gene) by heat shock
  • Thaw DH5-α competent bacteria in ice
  • 100 µL of bacteria + 10 µL ligation mix
  • Incubation 20 minutes in ice
  • 45 s at 42 ° C
  • 3 min in ice
  • Adding 900 μl of LB
  • Incubation 1 h, 37 °C
  • Spread on LB Ampicillin plates
  • Incubation overnight 37 °C
14th of August

No colony for PCT2 transformation!

Miniprep from precultures

Transformed bacteria were stored at -80 °C in 50 % glycerol (2 aliquotes for each preculture)

  • GenElute Plasmid Miniprep Kit (Sigma, PLN350-1KT)
  • Incubation 1 min for elution of DNA before centrifugation
  • pSB1A3-PhaC2 144 ng/µL
  • pSB1A3-PhaC3 96.1 ng/µL
  • pSB1A3-PCT2 143.3 ng/µL
Digestion

Mix

- LDH1 gBlock gene (10 ng/µL) pSB1A3 (X+P)
DNA 20 µL (200 ng) 50 µL
10X Buffer NEB CutSmart 3 µL 6 µL
enzyme XbaI 1 µL 2 µL
enzyme SpeI 1 µL -
H2O (qs 30 µL) 5 µL 2 µL (qs 60 µL)

Incubation 1 h 37 °C

Dephosphorylation
  • Add 1 µL alkaline phosphatase (FastAP #EF0654 Thermo Scientific) to the digestion mix (for LDH1 digestion or vector pSB1A3 digestion)
  • Incubation 30 min 37 °C
  • Inactivationof the enzyme: incubation 75 °C, 10 min
Recombinant vector digestion

Mix

- pSB1A3-PhaC2 pSB1A3-PhaC3 pSB1A3-PCT2
DNA (around 200 ng or 600 ng) 2 µL 2.5 µL 6 µL (around 600 ng)
Buffer NEB CutSmart 10X 3 µL (2.1) 3 µL (2.1) 3 µL (CutSmart)
Enzyme 1 1 µL (EcoRI) 1 µL (EcoRI) 1 µL (EcoRI)
Enzyme 2 1 µL (SpeI) 1 µL (SpeI) 1 µL (XbaI)
H2O (qs 20 µL) 14 µL 13.5 µL 10 µL

Incubation 1 h 37 °C

DNA purification (LDH gene)
  • DNA clean & concentrator-5 (#D4003S Zymo Research)
  • Ratio 5:1 of DNA binding buffer for LDH gene fragment
  • Elution 30 µL water with incubation 3 min before centrifugation
  • LDH1 =>
    • 6.9 ng/µL
Agarose gel for digested recombinant vectors

All of the digested samples is charged on the gel.

14-August_agarose-gel1

Digestion of the recombinant vector. Digestion was a failure for the 2 plasmids containing the PhaC2 and PhaC3 genes. The vector containing PCT2 is open but we have no way to be sure that the digestion was efficient for the 2 enzymes or just for one of them.

Colony PCR on the clones obtained after the 2nd transformation of the ligation mixes:

14-August_agarose-gel2

PCR colony. PCR on colony failed.


Week 7


16th of August

Digestion
- LDH1 gBlock gene (10 ng/µL) pSB1A3 (352 ng/µL)
DNA 30 µL (300 ng) 2 µL
10X Fastdigest buffer (ThermoScientific) 4 µL (clear buffer) 2 µL (green buffer)
enzyme XbaI 1 µL 1 µL
enzyme SpeI 1 µL 1 µL
H2O (qs 40 µL) 4 µL 14 µL (qs 20 µL)

- pSB1A3-PhaC2 pSB1A3-PhaC3 pSB1A3-PCT2
DNA (around 200 ng or 600 ng) 2 µL 2.5 µL 6 µL (around 600 ng)
Buffer Fastdigest green 10X (ThermoScientific) 2 µL 2 µL 2 µL
Enzyme 1 1 µL (EcoRI) 1 µL (EcoRI) 1 µL (EcoRI)
Enzyme 2 1 µL (SpeI) 1 µL (SpeI) 1 µL (XbaI)
H2O (qs 20 µL) 14 µL 13.5 µL 10 µL
Agarose gel 1 %
16-August_agarose-gel
DNA purification (digested by X + S LDH1 gene)
  • DNA clean & concentrator-5 (#D4003S Zymo Research)
  • Ratio 5:1 of DNA binding buffer for LDH gene fragment
17th of August

Gel purification via the « Zymoclean Gel DNA Recovery » kit
  • weight of the empty tube: 1 g
  • weight of the tube + vector PSB1A3 (X+S)
  • n°1 : 1.1 g is 100 mg
  • n°2 : 1.1 is 100 mg
  • vector size: 2155 bp
  • Step 1 : For 100 mg d’agarose we add 300 µL of ADB
  • Step 2 : Put samples at 55 °C for 10 minutes and centrifuge at 300 rpm
  • Step 3 : Transfert in the column
  • Step 4 : Centrifugation while 1 minute at 13 000 rpm then suppression of the flow-through
  • Step 5 : Add 200 µL of DNA Wash Buffer and centrifugation while 30 seconds X2
  • Step 6 : Centrifugation empty for 2 minutes
  • Step 7 : Add 10 µL DNA Elution Buffer, wait 2 minutes
  • Step 8 : Centrifuge 1 minutes at 13 000 rpm
Nano drop
  • DO vector Psb1a3 n°1 : 39 ng/µL
  • DO vector Psb1a3 n°2 : 43.2 ng/ µL
18th of August

Chloramphenicol stock
  • 50 ng/mL chloramphenicol stock (2.5 g in 50 mL ethanol 100 %)
  • 30 aliquots stored at -20 °C
pSB1C3-mRFP
  • Solubilisation of pSB1C3-mRFP (2016 IGEM plate 5 - 1F) with 10 µL water nuclease-free (ThermoScientific)
  • Incubation 5 min RT
  • Transformation in E. coli DH5-α strain
Transformation
  • Thaw 25 µL DH5-α competent bacteria in ice
  • Add 2 µL of resolubilized pSB1C3-mRFP DNA from 2016 IGEM plate 5
  • Incubation 20 minutes on ice
  • 1 min at 42 ° C
  • 3 min in ice
  • Adding 120 μl of LB
  • Incubation 1 h, 37 °C with shaking
  • Spread on LB Chloramphenicol plates
  • Incubation overnight 37 °C

Solubilisation of the primers iG051, iG052, iG049 and iG050 and aliquots of 10 times dilutions.

19th of August

Precultures

3 mL LB medium + 1.8 µL Chloramphenicol (50 mg/mL) => plates at 30 µg/mL chloramphenicol

  • No conclusive results for PCR on colonies for pSB1C3-mRFP plates so we did precultures with 4 other clones (A, B, C and D)
  • The Mastermix Taq DNA polymerase seems to not work anymore (after freezer problem, the polymerases lost their activities)
Q5 PCR for changing antibiotic resistance in pSEVA 224 plasmid
  • 10 µL reaction buffer
  • 1 µL dNTP mix
  • 1 µL 10 times diluted primers (X2)
  • 0.5 µL DNA template
  • 0.5 µL Q5 DNA polymerase
  • H20 qs 50 µL (= 36 µL)

2 different reactions:

  1. Amplification of spectinomycin resistance gene with pCDF vector as DNA template (length expected = 1220 bp) primers used: iG050 and iG052 (1 min of DNA polymerization and primers annealing at 60 °C)
  2. Amplification of pSEVA224 backbone without kanamycin resistance gene (pSEVA224 as DNA template; length expected = 4253 bp) primers used: iG049 and iG051 (3 min of DNA polymerization and primers annealing at 60 °C)

  • 95 °C 5 min
  • (95 °C 30 s, 60 °C 30 s, 72 °C 1 min or 3 min) * 35 times
  • 72 °C 5 min
  • 10 °C ∞
20th of August

DNA miniprep of pSB1C3-mRFP (clone A, B, C and D)
  • Stock in 50 % glycerol stored at -80 °C
  • GenElute Plasmid Miniprep Kit (Sigma, PLN350-1KT)
  • Incubation 2 min for DNA elution before centrifugation and centrifugation of 1 min 30 s instead of 1 min.
  • Optional Wash Buffer step performed and use of nuclease-free water for elution.
  • Clone A: 240.3 ng/µL
  • Clone B: 150.3 ng/µL
  • Clone C: 111.0 ng/µL
  • Clone D: 76.6 ng/µL
PCR on DNA miniprep - Primers iG001 and iG002

20 µL mix per sample with:

  • 10 µL Green Taq Master Mix 2X (#K1081 Thermo Scientific)
  • 0.25 µL Forward primer iG001 (10 µM)
  • 0.25 µL Forward primer iG002 (10 µM)
  • 9.5 µL H2O (qs 20 µL)

PCR program:

  • 95 °C 5 min
  • (95 °C 30 s, 50 °C 30 s, 72 °C 1 min) * 30 times
  • 72 °C 5 min
  • 10 °C ∞
Agarose gel 1 % for changing antibiotic PCR
20-August_agarose-gel

The amplification of pSEVA => new attempt

Digestion vectors pSB1C3-mRFP (on DNA miniprep)

  • 10 µL plasmid DNA
  • 3 µL Buffer 10X green FD (FastDigest)
  • 1 µL EcoRI
  • 1 µL SpeI
  • 15 µL H20 (qs 30 µL)

Size expected: 2051 bp

Digestion was checked on agarose gel and the band corresponding to pSB1C3 digested by E+S was cut in order to recover the DNA.

Week 8


22th of August

Digestion efficiency test of restriction enzymes EcoRI and SpeI

Mix

  • 4 µL vector pSB1C3
  • 3 µL buffer 10X CutSmart or FastDigest Green
  • 1 µL EcoRI and/or SpeI
  • 21 µL or 22 µL H2O

Incubation 1 h 37 °C

Agarose gel 1 %
22-August_agarose-gel
DNA extraction from agarose gel (linear vector extracted from clones A, B and C)
  • Zymoclean gel DNA Recovery kit #D4001S
  • Vector A: 20.9 ng/µL
  • Vector B: 13.3 ng/µL
  • Vector C: 13.1 ng/µL

Expected sizes after digestion by E+S

  • pSB1C3 2051 bp
  • mRFP 742 bp
23th of August

Digestion gBlocks by E+S
  • 20 µL gBlocks DNA
  • 3 µL 10X NEB Buffer (Cutmart)
  • 1 µL EcoRI
  • 1 µL SpeI
  • 5 µL H2O (qs 30 µL)
  • Incubation 1 h at 37 °C
Purification Agarose Gel with the kit « Zymoclean Gel DNA Recovery kit » (#D4001S Zymo Research)
  • Cf protocol august 17th 2016
  • Elution with 20µL H2O
Nanodrop 1
  • LDH2: 10.3 ng/µL
  • Phac3: 5.9 ng/µL
  • Phac 4: 8.5 ng/µL
  • Pct 2: 8.9 ng/µL

too low concentration for ligation so I put at the Speed Vae. Protocole "Vaccum Drive"; resuspend with 10 µL H2O

Nanodrop 2
  • LDH2 = 15 ng/µL - Ratio 260/280 : 1.92
  • Phac 3 = 8.8 ng/µL - Ratio 260/280 : 2.20
  • PhaC4 = 9.2 ng/µL - Ratio 260/280 : 1.05
  • Pct2 = 10.8 ng/µL - Ratio 260/280 : 1.88

Gene Length Amount (ng) Concentration (ng/µL) Volume (µL)
Plasmid psB1C3 2051 pb 20.9 2.4
PhaC3 1717 pb 209.3 8.8 23.8
LDH2 1227 pb 149.6 15 10
Phac 4 1720 pb 209.7 9.2 22.8
Pct 2 1612 pb 196.5 10.8 18.2

  • Amount of insert: 50*5 x length gBlock / length plasmid
  • Volume of insert: amount of insert / concentration of insert

As we suspended in 10 μl I do not have sufficient quantities.

Ligation mix


Insert DNA (µL) Vector (µL) Buffer T4 Ligase 10X (µL) T4 Ligase (µL) H2O (qs 20 µL)
Pct2 9 0.96 2 0.5 7.54
LDH2 9 1.29 2 0.5 7.21
Phac4 9 0.96 2 0.5 7.54
Phac3 9 0.96 2 0.5 7.54

  • I put at 22 °C the ligation Mix for 10 min
  • I take 5 µL for the transformation of 50 µL competent cells
Transformation
  • Use protocol of Bacterial transformation by heat shock
  • Thaw competent cells on ice
  • mixed 50 µL of competent bacteria with 5 µL of DNA
  • incubate on ice for 20 minutes
  • placing the tubes at 42 ° C for 1 min then put 3 min in ice
  • Add 250 µL of LB and incubate 1 h at 37 °C
  • Spread on box LB Chloramphenicol (30 µg/mL)
  • incubation overnight 37 °C
  • Poored 6 plates of 25 mL of LB agar + chloramphenicol (30 µg/µL)
  • Incubation at 37 °C
24th of August

Digestion gBlocks by E+S
  • 20 µL gBlocks DNA
  • 3 µL 10X NEB Buffer (Cutmart)
  • 1 µL EcoRI
  • 1 µL SpeI
  • 5 µL H2O (qs 30 µL)

Incubation 1 h at 37 °C

Purification Agarose Gel with the kit « Zymoclean Gel DNA Recovery kit » (#D4001S Zymo Research)

Elution with 20 µL nuclease-free water

  • PhaC3 (E+S) => 12.2 ng/µL
  • PhaC4 (E+S) => 6.3 ng/µL
  • LDH2 (E+S) => 16.8 ng/µL
  • PCT2 (E+S) => 12.4 ng/µL
  • Concentration of the DNA: around 1 h in evaporator
  • DNA resuspension with 11 µL water

First concentration failed so second concentration of DNA performed in evaporator (1 h)

25th of August

  • Resuspension of DNA with 11 µL water
  • PhaC3 (E+S) => 10.4 ng/µL
  • PhaC4 (E+S) => 10.6 ng/µL
  • LDH2 (E+S) => 27.0 ng/µL
  • PCT2 (E+S) => 20.1 ng/µL

DNA concentration by water evaporation failed!

Ligation genes inside pSB1C3 (ratio 5:1)

- PhaC4 PhaC3 PCT2 LDH2
Buffer T4 DNA ligase (10X) 2 µL 2 µL 2 µL 2 µL
T4 DNA ligase 0.5 µL 0.5 µL 0.5 µL 0.5 µL
Vector 0.96 µL 0.96 µL 0.96 µL 0.96 µL
Insert (gene) 7.9 µL 8.05 µL 3.91 µL 2.2 µL
H20 (qs 20 µL) 8.63 µL 8.49 µL 12.6 µL 14.3 µL

10 min incubation at 22 °C

Transformation
  • Thaw 50 µL bacteria (E. coli DH5-α) chemo-competent
  • Add 5 µL DNA (mix ligation)
  • Incubation 20 min on ice
  • Heat shock: 45 s at 42 °C
  • Incubation 3 min on ice
  • Add 950 µL LB
  • Incubation 1 h at 37 °C with shaking
  • Centrifugation 1 min at 4000 rpm
  • Throw away supernatant
  • Resuspension with the rest of supernatant (around 50 µL)
  • Spread on plates LB Chloramphenicol (30 µg/mL)
26th of August

Preparation plates LBC (30 µg/µL)

Ligation (ratio 1:1)

- PhaC4 PCT2 PhaC3 LDH2
Buffer T4 DNA ligase (10X) 1 µL 1 µL 1 µL 1 µL
T4 DNA ligase 0.5 µL 0.5 µL 0.5 µL 0.5 µL
Vector 2.39 µL 2.39 µL 2.39 µL 2.39 µL
Insert (gene) 3.96 µL 1.95 µL 4.02 µL 1.11 µL
H20 (qs 10 µL) 2.15 µL 4.15 µL 2.08 µL 5.0 µL

Incubation 16h 16 °C

28th of August

Transformation E. coli DH5-α
  • Thaw 25 µL chemo-competent bacteria
  • Add 5 µL DNA (mix ligation) (2 µL of pSB1C3-mRFP from clone A as positive transformation control)
  • Incubation 20 min on ice
  • Heat shock: 45 s at 42 °C
  • Incubation 3 min on ice
  • Add 900 µL LB
  • Incubation 1h20 at 37 °C with shaking
  • Centrifugation 1 min 30 s at 5000 rpm
  • Throw away all the supernatant
  • Resuspension with 100 µL LB medium
  • Spread on plates LB Chloramphenicol (30 µg/mL)
  • Incubation overnight 37 °C

Week 9


29th of August

The GFP gene contained in Gen4_BNR vector (provided by Cécile) was amplified by PCR with Q5 DNA polymerase with primers allowing to add the prefix and suffix regions at both sides of the amplified region.

Mix

  • 10 µL Q5 reaction buffer 5X
  • 1 µL dNTP mix
  • 2.5 µL of Forward Primer (iG063)
  • 2.5 µL of Reverse Primer (iG064)
  • 1 µL DNA template
  • 0.5 µL Q5 DNA polymerase
  • 32.5 µL Nuclease-free water (qs 50 µL)

PCR program:

  • 98 °C 30 s
  • (98 °C 30 s, 55 °C 30 s, 72 °C 1 min) 35 times
  • 72 °C 2 min
  • 10 °C ∞

After checking on agarose 1 % gel, we determined that the GFP was successfully amplified.

Digestion

The amplified fragment was digested by EcoRI and PstI during 1 h at 37 °C. The reaction was performed in NEB CutSmart buffer.

The digested mix was purified by using the kit Wizard SV Gel and PCR Clean-Up System (Ref: A9282, Promega).

30th of August

Ligation

The GFP fragment was then ligated separately in pSEVA 224 or pSEVA 2311 digested by E+P in a ratio 5:1.

The ligation mix was stored at -20 °C until transformation.

31th of August

September 2016


Week 9


2nd of September

Digestion

Genes Quantity of DNA (µL) Restriction Enzymes (µL) Buffer Water
Digest NEB 2.1 (µL) qs 50 µL (µL)
LDH2 (109.5 ng/µL) 10 EcoR1 HF : 1 µL 5 33
Pst1 :1 µL
PCT2 (56.5 ng/µL) 20 EcoR1 HF : 1 µL 5 23
Pst1 :1 µL
PhaC3 (63.9 ng/µL) 17.5 EcoR1 HF : 1 µL 5 25.5
Pst1 :1 µL
PhaC4 (88.1 ng/µL) 12.5 EcoR1 HF : 1µL 5 30.5
Pst1 :1µL
Vector pSB1C3 (240.3 ng/µL) 4.5 EcoR1 HF : 1 µL 5 38.5
Pst1 :1 µL

Incubation 1 h at 37 °C and after incubation 20 min at 80 °C.

Gel Extraction
  • 50 mL of Agarose 1 % with 2.5 µL of midorigreen.
  • Migration 25 min at 100 mV.
    • 2-September_agarose-gel

    *Digestion mixes with EcoRI and PstI were loaded on the gel.

3rd of September

DNA extraction from agarose gel

Kit: Wizard SV Gel and PCR Clean-Up System (Ref: A9282, Promega)

  • pSB1C3 => 6.4 ng/µL
  • LDH2 => 4.8 ng/µL
  • PCT2 => 2.9 ng/µL
  • PhaC3 => 4 ng/µL
  • PhaC4 => 5.8 ng/µL
DNA concentration by water evaporation
  • pSB1C3 => 7.1 ng/µL
  • LDH2 => 6.5 ng/µL
  • PCT2 => 5.5 ng/µL
  • PhaC3 => 4.0 ng/µL
  • PhaC4 => 5.0 ng/µL

DNA concentration still seems not efficient

DNA transformation
  • Thaw 50 µL chemo-competent DH5-α E. coli
  • Add 5 µL DNA (mix ligation with pSEVA 2311 or 224)
  • Incubation 25 min on ice
  • Heat shock: 45 s at 42 °C
  • Incubation 3 min on ice
  • Add 250 µL LB
  • Incubation 1h15 at 37 °C with shaking
  • Spread on plates LB Kanamycin (50 µg/mL) (50 µL of bacteria on one plate and 10 µL on another one for each vector)
  • Incubation overnight 37 °C
4th of September

Ligation in pSB1C3

- LDH2 (6.5 ng/µL) PCT2 (6.5 ng/µL) PhaC3 (4.0 ng/µL) PhaC4 (5.0 ng/µL)
Vector 4.2 µL 4.2 µL 4.2 µL 4.2 µL
Insert (gene) 2.8 µL 0.5 µL 0.5 µL 0.5 µL
Buffer T4 DNA ligase (10X) 2.39 µL 2.39 µL 2.39 µL 2.39 µL
T4 DNA ligase 3.96 µL 1.95 µL 4.02 µL 1.11 µL
H20 (qs 10 µL) 2.15 µL 4.15 µL 2.08 µL 5.0 µL

Incubation 1 h at 16 °C

Poor plates LBC (30 µg/mL chloramphenicol)

Transformation
  • Thaw 25 µL chemo-competent DH5-α E. coli
  • Add 4 µL DNA (mix ligation)
  • Incubation 25 min on ice
  • Heat shock: 45 s at 42 °C
  • Incubation 3 min on ice
  • Add 225 µL LB
  • Incubation 1 h at 37 °C with shaking
  • Spread 120 µL on plates LBC
  • Incubation overnight 37 °C

Week 10


7th of September

Digestion of genes

See complete version of the notebook (pdf) for details on the conditions.

Incubation 1 h at 37 °C and after incubation 20 min at 80 °C.

8th of September

Digestion of bacbone Igem

Mix: Enzyme Master Mix E+P

  • 0.5 µL NEB 2.1 Buffer
  • 0.5 µL BSA (Bovine Serum Albumin)
  • 0.5 µL EcoR1-HF
  • 0.5 µL Pst1
  • 0.5 µL Dnp1
  • 18 µL H2O

Mix of 4 µL of linearized backbone and 4 µL of master mix enzyme

Incubation of all the solution at 37 °C during 30 min and then, heat deactivation during 20 min at 80 °C.

Ligation

Genes length genes Volume Final Concentration Final amount for 1 µL (Cf/Vf)
pSB1C3 2037 bp 8 µL 100 ng 25 ng= 2 µL
LDH_V2 1227 bp 50 µL 1095 ng 21.9 ng/µL
PCT_V2 1612 bp 50 µL 1130 ng 22.6 ng/µL
PhaC_V3 1717 bp 50 µL 1118.25 ng 22.365 ng/µL
PhacC_V4 1720 bp 50 µL 1101.25 ng 22.025 ng/µL

Amount calculation of insert for Ligation with NEBio Calculator:


Genes Amount Volume for deduced
LDH_V2 15.06 ng 0.7 µL
PCT_V2 19.78 ng 0.9 µL
PhaC_V3 21.07 ng 0.9 µL
PhaC_V4 21.11 ng 0.96 µL

Mix of ligation

  • 2 µL of pSB1C3
  • Volume calculate of digested insert
  • 1 µL T4 Ligase Buffer
  • 0.5 µL T4 Ligase
  • qs 10 µL

I put the mix at 16 °C while 30 min and after heat kill for 20 min at 80 °C

Transformation by heat shock

With 2 µL of Ligation mix and 25 µL of Competent Cells.

  • 2 µL of Ligation with 25 µL of competent cells.
  • Put the mix 20 min in the ice.
  • Put the mix at 42 °C while 45 seconds
  • 2 min on the ice.
  • Add 950 µL of LB
  • Recovery 1 h at 37 °C with agitation
  • 7000 rpm during 1 min
  • Suppression of the flow-throw
  • Resuspend with the remaining
  • Spread on plates (put the simple in the middle of the plates)
  • Incubation overnight 37 °C
Plates preparation

1 plates = 25mL of LB Agar + 15 µL of chloramphenicol [30 µg/mL]

Preculture

3 mL LB liquid medium + 1 µL E. coli DH5-α bacteria from stock at -80 °C

9th of September

Preparation of chemo-competent E. coli DH5-α by heat shock
  • WORK STERILE
  • Launch a pre-culture of the strain in LB at 37 ° C
  • Inoculate 25 mL of LB 1/50 (500 μl) with the pre-culture
  • Let it growth at 37 ° C with stirring until OD(600 nm) = 0.5-0.7
  • After 1h30, OD= 0.9
  • For Spectrometer measurements, 1 mL of LB for the blank
  • A dilution was performed to obtain 0.5 OD(600 nm) and the culture was split into 2 falcons with 27.5 mL of culture
  • Cells are cooled 10 min on ice
  • Centrifugate culture 6 min at 4000 rpm at 4 °C
  • Suppression of the flow-through
  • Dilution of the pellet in 1/2 volume of cold CaCl2 0.1M (13.75 mL)
  • Cells cooled on ice during 20 min
  • Culture centrifugated 6 min at 4000 rpm at 4 °C
  • Suppression of the flow-through
  • Resuspension the pellet in 1 / 50e of final volume (550 μl final volume) of cold CaCl2 + 10 % glycerol
  • Vi= 110 µL
  • The mix is 440 µL CaCl2+ 110 µL glycerol 50 %
  • I multiplied by 2 the doses for have more mix
  • 22 aliquots of 50 µL bacteria
11th of September

PCR Amplification of LDH2, PCT2, PHAC3, PHAC4 with Q5 DNA polymerase

For 50 µL reaction

  • 25 µL of Q5 Master Mix
  • 2.5 µL of Forward Primer (IG063)
  • 2.5 µL of Reverse Primer (IG064)
  • 0.5 of Template DNA
  • 19.5 of H2O

PCR program

  • 98 °C 30 s
  • (98 °C 30 s, 55 °C 30 s, 72 °C 1 min) 35 times
  • 72 °C 2 min
  • 10 °C ∞

PCR Colonie LDH2 with One Taq

For 25 µL to 1 Reaction --> Vf= 125 µL

Mix

  • 25 µL of One Taq Standard Reaction Buffer
  • dNTPs = 2.5 µL
  • 2.5 µL of Forward Primer (IG001)
  • 2.5 µL of Reverse Primer (IG002)
  • 0.625 µL of One Taq DNA Polymerase
  • 91.9 µL of Nuclease Free Water

PCR program

  • 94 °C 30 s
  • (94 °C 30 s, 50 °C 1 min, 68 °C 1 min 45 sec) 30 times
  • 68 °C 5 min
  • 10 °C ∞

Week 11


12st of September

Miniprep colonies LDH
  • 3 µL of LB
  • Take one of the colony in the plates
  • Incubated at 37 °C overnight
Digestion Backbone IGEM pSB1C3 linearized by E+P

Mix: Enzyme Master Mix E+P for 22 reaction

  • 2 µL NEB 2.1 Buffer
  • 2 µL BSA (Bovine Serum Albumin)
  • 2 µL EcoRI-HF
  • 2 µL PstI
  • 2 µL DnpI
  • 72 µL H2O

  • 16 µL of linearized backbone and 16 µL of the MasterMix enzyme
  • Incubation at 37 °C during 30 min
  • Enzyme inactivation with 20 min incubation at 80 °C.
Precultures
  • 5 mL LB medium
  • + 5 µL antibiotic (Kanamycin 50 mg/mL for pSEVA 212S, Spectinomycin 50 mg/mL for pSEVA 424 and Gentamycin 10 mg/mL for pSEVA 628S)
  • + some bacteria (sent as bacteria aliquots in LB medium by de Lorenzo)
  • Incubation 37 °C overnight
13st of September

Miniprep of pSEVA plasmids

2 aliquots stocks in 50 % glycerol stored at -80 °C

GenElute Plasmid Miniprep Kit (Sigma, PLN350-1KT)

3 DNA vectors: pSEVA 424 (equivalent to the pSEVA 224 but with spectinomycin resistance gene instead of kanamycin, pSEVA 212S and pSEVA 628S used for genes deletion in genomic DNA in Pseudomonas putida)

Digestion of the “old” gene by E+P
Ratio 1 : 2
Genes Amount Volume for deduced
LDH_V2 30.12 ng 1.37 µL
PCT_V2 39.57 ng 1.71 µL
PhaC_V3 42.15 ng 1.88 µL
PhaC_V4 42.22 ng 1.92 µL

Ligation mix

  • 2 µL of pSB1C3
  • Volume calculate of digested insert
  • 1 µL T4 Ligase Buffer
  • 0.5 µL T4 Ligase
  • qs 10 µL

I put the mix at 16 °C while 30 min and after heat kill for 20 min at 80 °C

Minipreps DNA Purification System kit “Wizard Plus”
  • Take 2 µL of overnight culture
  • Centrifuge at top speed for 1 min
  • Suppression of the flow-throw
  • Resuspend pellet with 250 µL of Cell Resuspension Solution
  • Add 250 µL of Cell Lysis Solution and invert 4 times to mix
  • Add 10 µL of Alkaline Protease Solution and invert 4 time to mix
  • Incubate 5 min at room temperature
  • Add 350 µL of Neutralization Solution and invert 4 times to mix
  • Centrifuge at top speed for 10 min at room temperature
  • Insert Spin Column into Collection Tube
  • Decant cleared lysate into Spin Column
  • Centrifuge at top speed for 1 min
  • Discard flowthrough
  • Add 750 µL of Wash Solution and centrifuge at top speed 1 min
  • Discard flowthrough
  • Repeat with 250 µL of Wash Solution
  • Centrifuge at top speed for 2 min
  • Transfert Spin Column to 1.5 mL tube
  • Add 100 µL of Nuclease free water to the Spin Column
  • Centrifuge at the top speed for 1 min
  • Discard column

Ratio 1 : 5
Genes Amount Volume for deduced
LDH_V2 75.29 ng 3.44 µL
PCT_V2 98.92 ng 4.38 µL
PhaC_V3 105.4 ng 4.71 µL
PhaC_V4 105.5 ng 4.79 µL

Purification of Amplification PCR LDH2, PCT2, PHAC3, PHAC4
Nanodrop

For the genes


Genes Concentration (ng/µL) Ratio 260/280
LDH2 62.7 1.78
PCT2 52.8 1.79
PHAC3 50.3 1.76
PHAC4 69.7 1.78

For the LDH colonies


Colonies Concentration (ng/µL) Ratio 260/280
LDH_n°1 70 1.92
LDH_n°2 31 2.06

Digestion “New” genes LDH2, PCT2, PHAC3, PHAC4

Digestion by EcoR1-HF+PSt1


Genes Quantity of DNA (µL) Restriction Enzymes Buffer Digest NEB 2.1 (µL) H2O
LDH2 (62.7 ng/µL) 16 1 µL EcoR1-HF 5 27
1 µL Pst1
PCT2 (52.8 ng/µL) 19 1 µL EcoR1-HF 5 24
1 µL Pst1
PHAC3 (63.9 ng/µL) 16 1 µL EcoR1-HF 5 27
1µL Pst1
PhAC4 (69.7 ng/µL) 14.5 1 µL EcoR1-HF 5 28.5
1 µL Pst1
pSB1C3 (240.3 ng/µL) 4.5 1 µL EcoR1-HF 5 38.5
1 µL Pst1

Other digestion


Genes Quantity of DNA (µL) Restriction Enzymes Buffer Digesr NEB 2.1 (µL) H2O
PHAC3 16 1 µL Xba1 5 27
1 µL Pst1
PHAC4 14.5 1 µL Xba1 5 28.5
1 µL Pst1
PCT2 19 1 µL EcoR1-HF 5 24
1 µL Spe1

Incubation at 37 °C during 30 min and then, enzymes deactivation by heat kill for 20 min at 80 °C.

PCR Purification
Nanodrop Vf = 50 µL

Genes Concentration (ng/µL) Ratio 260/280
LDH_V2 62.7 1.78
PCT_V2 52.8 1.79
PHAC_V3 50.3 1.76
PHAC_V4 69.7 1.78

14st of September

The promoter region of the vectors pSEVA 224 and pSEVA 2311 were amplified by PCR with Q5 DNA polymerase with primers allowing to add the prefix and suffix regions at both sides of the amplified region.

Mix

  • 10 µL Q5 reaction buffer 5X
  • 1 µL dNTP mix
  • 2.5 µL 10 µM Forward Primer (iG063)
  • 2.5 µL 10 µM Reverse Primer (iG064)
  • 1 µL DNA template
  • 0.5 µL Q5 DNA polymerase
  • 32.5 µL Nuclease-free water (qs 50 µL)

PCR program:

  • 98 °C 30 s
  • (98 °C 30 s, 55 °C 30 s, 72 °C 1 min) 35 times
  • 72 °C 2 min
  • 10 °C ∞

After checking on agarose 1 % gel, we determined that the fragments were successfully amplified.

Digestion

The amplified fragments were digested by EcoRI and PstI during 1 h at 37 °C. The reaction was performed in NEB CutSmart buffer.

The digested mixes were purified by using the kit Wizard SV Gel and PCR Clean-Up System (Ref: A9282, Promega).

Design Primer for Gibson Assembly -> PR_IG0075 -> 81

15st of September

Ligation

The 2 fragments were then ligated separately in linear pSB1C3 provided by IGEM Headquarters in a ratio 5:1.

The ligation mix was used for transformation in E. coli DH5-alpha.


Transformation

  • Thaw 25 µL chemo-competent DH5-alpha E. coli
  • Add 3 µL DNA (mix ligation)
  • Incubation 20 min on ice
  • Heat shock: 45 s at 42 °C
  • Incubation 3 min on ice
  • Add 225 µL of LB medium
  • Incubation 1 h at 37 °C with shaking
  • Spread 120 µL on plates LBC
  • Incubation overnight 37 °C

    • 16th of September

    PCR colony

    Mix (25 µL total volume reaction):

  • 12.5 µL DreamTaq PCR Mastermix 2X (ThermoScientific)
  • 2.5 µL 10X DreamTaq Green Buffer (ThermoScientific)
  • 0.25 µL Forward Primer (0.5 µM) iG001
  • 0.25 µL Reverse Primer (0.5 µM) iG002
  • 9.5 µL H2O filtrated (qs 25 µL)

    • + clones


    Agarose gel 1 %

    16-September_agarose-gel

    Precultures

    Positive clones selected from the agarose gel results were picked and used to inoculate 3 mL of LBC (25 µg/mL chloramphenicol) medium.


    Observation

    All the precultures have grown


    PCR colony

    Mix (25 µL total volume reaction):

  • 10 µL DreamTaq PCR Mastermix 2X (ThermoScientific)
  • 1.5 µL Forward Primer (0.5 µM) iG001
  • 1.5 µL Reverse Primer (0.5 µM) iG002
  • 6 µL H2O filtrated (qs 25 µL)

    • + clones


    Migration map

    16-September_map-gel
    16-September_map-gel2


    October 2016


    Gel shown the last results corresponding to the genes selected

    The promoters isolated from vectors pSEVA2311 and pSEVA224 were also sent to iGEM HQ after checking by sequencing.

    20-October_gel

    For information, PhaC3 and PCT2 without RBS were successfully cloned in pSB1C3 and sent to iGEM HQ (verified by sequencing)



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