Paris/August 22
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The 96 well plate is surrounded by H<sub>2</sub>O 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 96 well plate is surrounded by H<sub>2</sub>O 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. | The growth profile is measured over a period of 20H, a DO measurement is acquired each 4min10s. | ||
| - | [[Results | + | [[Results Growth kinetics 220807|Results]] |
== Preparing chemically competent cells == | == Preparing chemically competent cells == | ||
Revision as of 00:18, 27 October 2007
Contents |
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
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.
