Team:Paris Bettencourt/MultiHost

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Revision as of 08:48, 29 August 2011

Team IGEM Paris 2011

Electro-poration protocol for B. subtilis

Electroporation protocol adapted from Cao et al. article (2011) that has given us some stable results over time. Because of the resistance of Bacillus subtilis to electro-poration higher voltages are needed to the point of destroying the population, all the different sugars used here are meant to stabilize the membrane of the cells.

Reagents and Equipment needed

Mannitol, Sorbitol, Trehalose, LB, glycerol (99,5%)
DNA (50 ng/μL), B. subtilis strain for transformation (no need to be competent)
Cuvette (2mm), Gene Pulser (Bio-rad) set on 200 ohms and 25 μF (≈ 5 ms pulses) and between 2 and 2.5 kV
Centrifuge set at 3000g and 10 minutes
Micropipettes: P2, P200, P1000
Pipettes: 25 mL, 10 mL, 5 mL
Polyethylene round bottom tubes

Day 1: preparation

Growth medium:
- LB + 0.5 mol.L^-1 sorbitol → expected final volume: 52 mL for 1 cell culture (including 1 mL for taring the absorbance machine)
  - msorbitol ≈ 4,736 g
Electro-poration medium:
- de-ionized water + 0.5 mol.L^-1 sorbitol + 0.5 mol.L^-1 mannitol + 0.5 mol.L^-1 trehalose + 10% glycerol (v/v) → expected final volume ≈ 40 mL for 1 round of poration
  - m_sorbitol = m_mannitol ≈ 3,643 g
  - m_trehalose ≈ 7,566 g
  - V_{99,5 % glycerol} ≈ 4 mL
Recovery medium:
- LB + 0.5 mol.L^-1 sorbitol + 0.38 mol.L^-1 mannitol → expected final volume: ≈ 1.1 ml per poly... tube (Approximately 10 tubes ≈ 11 mL total)
  - m_sorbitol ≈ 1,002 g (for 10 tubes)
  - m_mannitol ≈ 0,761 g (for 10 tubes)
Sterilise the solution: Filtration or autoclave.
Inoculate a falcon containing 10 ml of LB with your subtilis strain and let it grow overnight (37°C with shaking).

Day 2: electro-poration

Monitor the OD600 of your overnight culture.
In a 500 ml erlenmeyer: dilute your culture into 50mL of Growth Medium so that the OD 600 is 0.01.
Let the culture grow (37°C with shaking) until OD600 is between 0.85 and 1.
Cool the cells on ice for 5 minutes.
NOTE: KEEP ALL YOUR MATERIAL ON ICE AND ALWAYS MANIPULATE ON ICE FROM NOW ON, KEEP AS STERILE AS POSSIBLE.
Distribute evenly the culture into two falcons and centrifuge at 3000g for 10 minutes.
Get rid of supernatant, tap the falcon upside down on a piece of paper to get rid of as much solution possible. Detach the pellet.
Add 20 mL of ice-cold electro-poration medium to one falcon, suspend the cells and transfer the content to the other falcon. Re-suspend.
Centrifuge 3000g for 10 minutes.
Remove supernatant, detach pellet, add 10 mL of ice-cold electro-poration medium. Centrifuge.
Repeat step 10 with 5 mL, 2.5 mL and finally add 0.625 mL (1/80 of initial volume).
During the centrifugation time, prepare the polyethylene tubes (label them) with recovery medium in them and put the cuvettes on ice: 1 of each at least has to be a control of cells without DNA, then 1 for each transformant you wish to make.
Transfer in a cuvette: 60 μL of cells + 1 μL of DNA (50ng/μL; none if control).
Pulse the cuvette.
Transfer immediately the content into the poly... tube (STERILE CONDITIONS).
Repeat 13, 14 and 15 for the number of prepared cuvettes.
Incubate the polyethylene tubes at 37°C for 3 to 6 hours.
Prepare plates with antibiotics (none for the control).
Note: ≈ 25 ml of LBA per petri dish, make sure the antibiotic is well diluted, labeling should be obvious.
Plate max 100 μL of transformed cells per petri dish and let grow overnight. More is going to create a biofilm if the transformation worked and therefore make it impossible to select a single colony.

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 *Inoculate a falcon containing 10 ml of LB with your ''subtilis'' strain and let it grow overnight (37°C with shaking).
 <br>
+----
 ===Day 2: electro-poration===