Paris/August 22

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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.

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