Paris/August 22


yesterday -- tomorrow


Plasmids: MiniPrep Products

Plasmids: MiniPrep Products
Number Name Description Products in use Date
L31 Lox71>>B0015 Terminator Ligation of B0015 Terminator behind Lox71 L31.1, L31.3, L31.4 August 20
L32 B0015 Terminator>>Lox66 Ligation of B0015 Terminator before Lox66 L32.1, L32.2, L32.3 August 20
L34 Lox71-GFP-Ter dans J61002 L34.1, L34.2, L34.3 August 20
L35 AraC pBAD >> Lox71-GFP-Ter L35.1, L35.2, L35.3 August 20
L36 Lox66 >> mRFP L36.1, L36.2, L36.3 August 20
L37 Lox66 >> RBS-DapA Coli L37.1, L37.2, L37.3 August 20
L39 RBS DapA Coli BB dans pSB1A2 L39.1, L39.2, L39.3 August 20
L40 RBS DapA Subtilis BB dans pSB1A2 L40.1, L40.2 August 20
L41 CRE ORF >> lox71 FtsZ dans pSB1A2 (D47) L41.1 August 20
L42 CRE ORF >> lox71 FtsZ+A dans pSB1A2 (D47) L42.1, L42.2, L42.3 August 20
L43 RBS-DapA Subtilis FI >> CRE ORF dans pSB1A2 (D48) L43.1, L43.2, L43.3 August 20

Strains (glycerol stock)

Strains glycerol stock
Number Name Description Verified clone Date
S34 L31 Insertion of B0015 terminator behind lox71 August 22
S35 L32 Insertion of B0015 terminator in front of lox66 August 22
S36 L34 pTet>>lox71-gfptripart August 22
S37 L35 araC/pBad>>lox71-gfptripart August 22
S38 L36 lox66-mRFP August 22
S39 L37 lox66-RBS-dapAcoli BI August 22
S40 L39 RBS-dapAcoli cloned in pSB1A2 August 22
S41 L40 RBS-dapAsubtilis cloned in pSB1A2 August 22
S42 L41 lox71-ftsZ cloned in pSB1A2 August 22
S43 L42 lox71-ftsA-Z cloned in pSB1A2 August 22
S44 L43 RBS-dapAsubtilis cloned in pSB1A2 August 22

Growth kinetic on w121 strains

In order to precise the growth behavior/kinetics of our strain W121 in smaller DAP concentration, we fit those last ones in function of our last kinetics results (06/08/07)

Kinetic Array :w121 kinetic as a function of DAP and supplemented medium (S0.x)
1 2 3 4 5 6 7 8 9 10 11 12
A H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20
B H20 LB+0µM DAP LB+5µM DAP LB+8µM DAP LB+10µM DAP LB+12µM DAP LB+15µM DAP LB+17µM DAP LB+20µM DAP LB+30µM DAP LB+40µM DAP H20
C H20 S0.2+0µM DAP S0.2+5µM DAP S0.2+6µM DAP S0.2+7µM DAP S0.2+8µM DAP S0.2+9µM DAP S0.2+10µM DAP S0.2+15µM DAP S0.2+17µM DAP S0.2+20µM DAP H20
D H20 S0.4+0µM DAP S0.4+2µM DAP S0.4+3µM DAP S0.4+4µM DAP S0.4+5µM DAP S0.4+8µM DAP S0.4+10µM DAP S0.4+12µM DAP S0.4+15µM DAP S0.4+20µM DAP H20
E H20 S0.6+0µM DAP S0.6+1µM DAP S0.6+2µM DAP S0.6+3µM DAP S0.6+4µM DAP S0.6+5µM DAP S0.6+6µM DAP S0.6+7µM DAP S0.6+8µM DAP S0.6+10µM DAP H20
F H20 S0.8+0µM DAP S0.8+0,5µM DAP S0.8+1µM DAP S0.8+2µM DAP S0.8+3µM DAP S0.8+4µM DAP S0.8+5µM DAP S0.8+6µM DAP S0.8+7µM DAP S0.8+10µM DAP H20
G H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20
H H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20 H20

The 96 well plate is surrounded by H2O in order to maintain humidity during the assay. In each slot, 200µL of the growth medium (LB or S0.x) is mixed with 2µL of w121 culture grown ON, and with different amount of DAP (see table). The growth profile is measured over a period of 20H, a DO measurement is acquired each 4min10s. Results

Here are the results from our growth kinetic expereriment with limit DAP concentrations.

Preparing chemically competent cells

  • w121
  • FX85

1. Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
2. Grow the diluted culture to an OD600 of 0.5.
3. Put 500µL eppendorf tubes on ice so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled.
4. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
5. Centrifuge for 10 minutes at 3000 rpm and 4°C.
6. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media. Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
7. Add 100 μl aliquots to your chilled eppendorfs and store at − 80°C.

For TSS preparation (to make 50 mL) :

  • 5g PEG 8000
  • 0.30g MgCl2*6H20
  • 2.5 mL DMSO
  • Add LB to 50 mL

Filter sterilize (0.22 μm filter)
Store at 4°C or -20°C

Transformation of W121 and FX85 strains

  • W121 (DapA-) transformed with L30A & L30B (DapAp >> mRFP)
  • FX85 (lox-kan-lox) transformed with L26.1 & L26.2 (AraC pBAD >> CRE)

1. Thaw TSS cells on ice.
2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
3. Let sit for 30 minutes on ice.
4. Incubate cells for 30 seconds at 42°C.
5. Incubate cells on ice for 2 min.
6. Add 1 mL LB at room temp.
7. Incubate for 1 hour at 37°C on shaker.

For W121 strains:
8. Spread 200µL and 20µL onto different plates made with Ampicillin and DAP !.
9. Grow overnight at 37 °C.

For FX85 strains, see the recombination rate test chapter please.

Recombination Rate Test

For FX85 strains:
8. Spread 100µL of 1mL onto plates made with Ampicillin and THYMINE 0,02% !. Let grow overnight at 37°C
9. Take the rest (900 µL) and spread it into 100mL of LB (AMP-THYMINE 0,02%). We made 4 different Arabinose concentrations (0mM, 10µM, 1mM and 100mM).
10. The following day, count colonies on the plates and retake 100 of them onto a plate with AMP-KAN-THYMINE 0?02% !!!. Dillute to 10-7 the liquid culture and spread 100µL of the dillution prep to plates with Amp-Thymine 0,02%.
11. The day after, count how many of the 100 colonies have grown on AMP-KAN. Retake 100 clones from the plates made with the 10-7 dillution of the liquid culture. Spread them on AMP-KAN-THYMINE 0,02%.
12. Finally count how many clones have grown. This gives the percentage of clones whom the CRE was expressed and recombined the lox-kan-lox chromosome site in FX85 strains.