Ferment It Yourself

iGEM Paris-Bettencourt 2O15

Analytical digestion protocol

Prepare the following mix:
- 2µL 10X Digestion buffer
- 0.5µL Eco31I
- 0.5µL BbsI
- 2µL of DNA (200ng)
- 15µL water
incubate 1h at 37°C

Annealing Protocol

Phosphorylation of the oligos

5.6μL DNAse/RNAse free water
6.0μL oligo 1 (10µM)
6.0μL oligo 2 (10µM)
2.0μL 10X T4 DNA ligase buffer
0.4μL T4 PolyNucleotide Kinase

Total: 20μL

incubate 30min at 37°C
add 1μL of 1M NaCl
incube 5min at 95°C
let the mix cool down
use 2μL of the mix as a 10X solution

Chemical test for competent cells

20-30 sec at 8-10 krpm for the DNA tube (from the kit).
Thow cell competent on ice. Label 2 ml eppendorf µtube for each concentration and put it on ice.
Add 1µl of DNA into each µtube.
Add 50µl of competent cell into each tube. Flick gently to mix. Incubate in ice for 30 minutes. Pre-heat waterbath at 42°C.
Heat-shock cell by placing in waterbath 1 minute. CAUTION : The top need to be close to the water level, but not immerge in.
Keep back the tube to ice for 5 minutes.
Add 200µl of SOC media, incubate 37°C for 2h. Prepare agar plate of the media that you want, and label it (3 plate for each concentration).
Add 20µl of each tube n the appropriate plate. Incubate 37°C for OVN (12-16h).

Electroporation

Thaw electrocompetent cells on ice
Add 2µL of ligation product or 0.5µL of native plasmid to the cells
Transfer the cells in an 0.2mm electroporation cuvette
put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF
add 200µL of LB right after pulsing
recover 2 hours at 37°C
plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C

Electrocompetent Cells

Inoculate a 250mL LB flasks with 2.5mL of an overnight culture of cells
Incubate until the the DO600 reach 0.6 to 0.8
Place the cultures on ice for 15 minutes
Pour the culture in cold sterile 50mL falcon tubes
Centrifuge for 10 minutes at 4000rpm, 4°C
Throw the supernatant
Resuspend the cells in 50mL cold distilled water
Place the cells on ice for 10 minutes
Centrifuge them for 10 minutes at 4000rpm, 4°C
Throw the supernatant
Resuspend the cells in 12.5mL cold 10% glycerol
Place the cells on ice for 10 minutes
Centrifuge them for 10 minutes at 4000rpm, 4°C
Throw the supernatant
Resuspend the cells in 5mL cold 10% glycerol
Make aliquots of 50 to 100µL in microcentrifuge tubes and freeze them at -80°C

Digestion

Prepare the following mix:
- 4μL of Enzyme 1 Fast Digest
- 4μL of Enzyme 2 Fast Digest
- 4μL of FastAP
- 12μL of Fast Digest buffer 10X
- 1 to 3 μg of DNA
- up to 120μL of water
mix by pipetting up and down
incube 10 to 20 minutes at 37°C
incube 10 minutes at 68°C to inactivate the enzymes

Lactobacillus plantarum electrocompetent cells

Inoculate 5ml MRS medium with L. plantarum freezer stock.
Grow overnight at 30°C without shaking.
Add 1.25g glycine to two flasks of 50ml MRS medium.
Shake to dissolve.
Add 1ml overnight culture to each flask (1:50 dlution).
Culture for ~3 hours at 37°C with shaking.
Centrifuge culture for 5min at 4000g
Resuspend in 25ml ice-cold DI water.
Repeat it.
Centrifuge for 5min at 4000g
Resuspend in 5ml 50mM EDTA.
Incubate on ice for 5 minutes
Add 25ml ice-cold DI water.
Centrifuge culture for 5min at 4000g
Resuspend in 25ml ice-cold DI water.
Centrifuge culture for 5min at 4000g
Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
Repeat it.
Centrifuge culture for 5min at 4000g
Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
Keep on ice until use (within the next two hours).
Add 10μL of plasmid DNA to the 90μL of cell concentrate.
Keep on ice for 5 minutes.
Put 1mm cuvettes on ice too.
Pipette the cell/DNA mixture into the cuvette
Electrporate at 1200 volts
Time constant should be ~5.0
Immediately transfer the electroporated cells to 900μL of MRS medium.
Incubate at 30°C for 2-3 hours.
Plate on medium with the appropriate antibiotic.
Incubate at 30°C for two days.
Pick a colony

Heat Shock Transformation

Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min.
add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C.
put the cells back on ice for 2min.
add 200µL of LB to the cells and incubate 2 hours at 37°C.
plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.

Vitamin A Titration

Take 1g of food (with particle < 1mm of diameter) in bottle protected of the light, and add 15ml of hexane. Mix it.
Add again 15ml of hexane and shake during 5-10 minutes (do the same time, if you want compare two food sample).
In an other bottle protected of the light, do a filtration (coarse cellulose filter) of the previous solution.
Take the batter who don't pass the filter, and 15ml of hexane, shake 5 minutes, and filtered it like previously, to take all B-caroten as possible from the food sample.
Put in the fridge at -20°C to conserve it.
Obtain the result with a spectrophotometer with wavelenght of 450 nm (with a blank of hexane).

Vitamin A Titration using HPLC

Suspend cells in 1 mL of sterile water.
Add 0.5 to 0.75 mm glass beads and vortex for 3 minutes.
Add 2.5 mL of 0.2% (wt/vol) pyrogallol dissolved in methanol and vortex for 3 minutes.
Add 1.25 mL of 60% (wt/vol) KOH and vortex for 10 seconds.
Incubate for 1 h at 75ºC, vortexing every 15 minutes.
Add appropriate amount of hexane.
Centrifuge tubes for 5 minutes at 2,800 rpm.
Pipette 1 mL of hexane into a cuvette.

Lactococcus lactiselectrocompetent preparation

Prepare the following solutions

Glycine/Sucrose solution

80mL of 50% sucrose solution
29.2mL of 20% glycine solution
7.6mL of water

Electroporation buffer
- 0.125g of NaHPO₄
- 34.2mL of 50% sucrose solution
- 100µL of MgCl₂ 1M
- 65.5mL of water
Electroporation buffer + glycreol
- 187.5µL of 80% glycerol
- 812.5µL of electroporation buffer

Protocol:

Inoculate 10mL of M17 + 1% glucose (GM17) with some Lactococcus and incubate overnight at 30°C
Inoculate a 100mL flask of GM17 with the 10mL of overnight culture
Incubate the flask at 30°C until DO reach 0.4 to 0.5
Add 100mL of the glycine/sucrose solution
Incubate 1 hour at 30°C with shaking
Split the 100mL culture in twyo 50mL centrifuge tubes
Centrifuge the cells 10 minutes at 6000 rpm
Throw the supernatant and work on ice for the rest of the protocol
Resuspend the cells in 20mL of electroporation buffer
Centrifuge the cells 10 minutes at 6000 rpm and then throw the supernatant
Resuspend the cells in 20mL of electroporation buffer
Centrifuge the cells 10 minutes at 6000 rpm and then throw the supernatant
Resuspend the cells in 0.5mL of electroporation buffer
Centrifuge the cells 10 minutes at 6000 rpm and then throw the supernatant
Resuspend the cells in 1mL of cold electroporation buffer + glycerol
Aliquots the cells in eppendorf tubes
Save the cells at -80°C

Yeast Lysis with NaOH

20 µl NaOH into PCR tubes
Pick colonies into NaOH
Incubate at 95°C for ~45 minutes
Centrifugate at 8000 krpm for 10 minutes
Use 1 µl supernatant as template in a (10 µl) PCR

Heat Shock Transformation for Yeast

After growth, determine the titer of the yeast culture by using spectrophotometer : pipette 10µL of cells into 1mL of water in spectrophotometer cuvette and measure the OD at 600nm.
Add 2.5x10⁸ cells to 50mL of 2X YPD in a culture flask.
Incubate the flask in a shaking incubator at 30°C until the cell culture is at least 2x10⁷ cells.mL^-1
Denature 1mL of carrier DNA at 99°C for 5min and chill immediately in ice.
Harvest the yeast cells by centrifugation at 3,000g for 5min.
Resuspend the pellet in 25mL of sterile water and centrifuge at 3,000g for 5min at 20°C. Repeat this wash with sterile water 2 times.
Resuspend the last pellet in 1mL of sterile water.
Transfer the cell suspension to a 1.5mL microcentrifuge tube.
Centrifuge for 30s at 13,000g and discard the supernatant.
Resuspend the cells in 1mL of sterile water and pipette 100µL samples into 1.5mL microcentrifuge tubes, one for each transformation.
For each transformation :

240µL of PEG 3350 (50% (w/v))
36µL of LiAc 1.0M
50µL of single-stranded carrier DNA (2.0mg.mL^-1)
6µL of PCR product
28µL of water DNAse Free

Place the tubes at 42°C for 40min.
Centrifuge the tubes at 13,000g for 30s in a microcentrifuge tube and remove the supernatant.
Pipette 1mL of YPD liquid medium into the transformation tube, and vortex mix to resuspend pellet.
Incubate 3h at 30°C to ensure good antibiotic expression.
Plate 2, 20 and 200µL of the cell suspension onto YPD medium with 200µm.mL^-1 antibiotic G418.
Incubate the plates at 30°C for 3 days.

Ligation

Mix the following:
- vector 100ng
- insert 300ng
- 2µL T4 DNA ligase buffer 10X
- 1µL T4 DNA ligase
- up to 20µL of water
incubate 10 to 15 minutes at 22°C
incubate 5 minutes at 70°C

Vitamin B2 Titration using HPLC

Idli Recipe

Soak rice and dall separately for 4-5 hours. (for 1.5 volume of rice, put the same volume of water, and 0.5 volume of dall, put the same volume of water).
Rinse both, mix it, add 2/3 volume of water of this volume.
Blend this mix, and let ferment for 12-16 hours.
Then Cook it with idli cooker for 15 minutes.

In the case where we add micro-organisms, we have 3 options:

Add µorganisms during the soak phase
Add µorganisms after the blending phase.
Let soak µorganisms in the same time that rices and dall, and add it for the blending phase.

The third was mainly used.

Analytical digestion