Team:Paris Liliane Bettencourt/Notebook/2011/08/02/

From 2011.igem.org

Team IGEM Paris 2011

Adrien Lhomme-Duchadeuil

First, an estimation of how much antibiotics is necessary for Bacillus subtilis (thank you Axel).
Appropriate concentration in antibiotics

Chloramphemicol : 5-10 µg/mL
Spectinomycin : 50-100 µg/mL
Tetracyclin: around 75 µg/mL
Kanamycin: 5 µg/mL
Erythromycin: 1 µg/mL + 25 µg/mL lincomycin

Results of electroporation: IT WORKED after three trials!

Positive control worked
Negative controls didn't show any colonies except when put in the presence of TetR (qty:12,5 µg/mL or 5µg/mL) which was insufficient => I will re-plate the improbable survivors on plates of different tetracyclin concentrations to test resistance.
Cm resistant strain have survived in low quantities at both 30 and 5 µg/mL of antibiotics
It seems that even cuvettes that had an electric arc were able to produce a couple colonies, which means that it doesn't anihilate all the bacteria.

Table of resutls of electroporation using Cao et al. method
tube 1: poration control no DNA (12,5 kV: ARC) CTL ⁺: carpet CTL ^- with Cm (5 and 30 µg/mL): none CTL ^- with TetR (5 and 12,5 µg/mL): carpet	tube 2: poration control no DNA (10kV: OK) CTL ⁺: carpet CTL ^- with Cm: none CTL ^- with TetR: carpet	tube 3: pHM3::TetR (12,5 kV: OK) TetR(5 and 12,5 µg/mL): carpet	tube 4: pHM3::TetR (10 kV: OK) TetR (12,5 µg/mL): carpet	tube5: pHM3::TetR (12,5 kV: ARC) TetR (5 and 12,5 µg/mL): carpet	tube 6: pHM3::TetR (10 kV: OK) TetR (5 µg/mL): carpet
tube 7: S12::Cm (12,5 kV: ARC) Cm 5 µg/mL: 1 colony Cm 30 µg/mL: 1 colony	tube 8: S12::Cm (10 kV: OK) Cm 30 µg/mL: 4 colonies	tube 9: S12::Cm (12,5 kV: OK) Cm 30 µg/mL: 9 colonies Cm 5 µg/mL: 4 colonies	tube 10: S12::Cm (10 kV: OK) Cm 5 µg/mL: 1 colonie	CTL ^--: no bacteria no growth

Transformation of B. subtilis via electro-poration based on Cao et al. 2011 article

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 2 or 2.5 kV
Centrifuge set at 3000g and 10 minutes
Micropipettes: P2, P200, P1000
Pipettes: 25 mL, 10 mL, 5 mL

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 poly... 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 poly... 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 150 μL of transformed cells per petri dish and let grow overnight.

Axel

Transformation in B. subtilis

With fresh culture of subtilis grown in MDCH medium, make transformation of it with k143079. When plating, play a bit with concentration of chloramphenicol (from 5 to 15 µg/mL)

Cyrille

Transformation of the QCM of pHM3

First the transformation of the quick change done yesterday was done using both MH1 and commercial competent cells, directly, and then 20 µL and 200µL plated on ampicilin plates (done the same day). This is a mistake from my side, because I forgot to gigest by DpnI before doing the transformating.

Hopefully, there where some PCR products left. So I digest the rest of the PCR product with 1,2 µL of DpnI during 20 min and then I did the transformation again with MH1 cells, and plated.

We will see the result tomorow. A gel was done after the digestion by DpnI. I did the things fast, so that one well was pierced and I lost the DNA. The result on the gel is the following. XX ng means the amount of DNA that was given as a template.

DNA ladder - Negative control with DNA digested - 10 ng - 50 ng (lost) - 100 ng

We see that the DNA template is not visible on the gel whereas there are two thin visible bands on the gel at the good size. The enligtment at the end of the gel is due to the DpnI digestion bands.

Well the results seems correct, but only the final insulated clones will say the result.

Sequencing

We prepared the tubes but we missed the transporter. They will be taken tomorow. We will have to wait one day more, sorry...

Send to garantee the PRC machine

I called the maintenance center and packed the PCR machine for the transporter to take it tomorow... Things to follow.

Kevin

Culture of double transformed cells (suite)

Diluted at 1/100 => expected to be between OD 300 and 600

3 Tubes pSG20 at 37°C
3 Tubes pFX234 at 37°C

3 Tubes pSG20 only at 30°C
3 Tubes FX234 only at 30°C
3 Tubes pSG20/pDAG464 at 30°C
3 Tubes pFX234/pDAG479 at 30°C

At OD 300-600, induction of fusion protein with arabinose 0%, 0,1 % and 0,2% for 1h30.

Microscopy (suite)

Old Zeiss doesn't work, we have to do everything again tomorrow.

Camille & Danyel

We did minipreps of our clons from the day before and glycerols.