Team:Paris/August 22

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Contents

1 Analysis of the transformant of FlhDC+promotor
2 Construction of pFlgA - GFP Generator
- 2.1 Digestion
- 2.2 Ligation
3 Construction for FIFO
- 3.1 Transformation of the ligations we did yesterday
4 Construction for synchronization
5 Promoter characterization plasmids
- 5.1 Transformation of ligations from August 20th

Analysis of the transformant of FlhDC+promotor

Analysis of the plasmids MP160.1 and MP160.2 (FlhDC+promotor in pSB1A2)
Control: MP143 (GFP generator in pSB1A2)

PCR

PCR screening programm
elongation time: 1 min 30
total volume reaction (25 µL)

12,5 µL Quick load PCR Mix 2X
0,5 µL O18
0,5 µL O19
1 µL DNA
10,5 µL water

Digestion

total volume reaction (30 µL)

2 µL DNA
3 µL buffer 2 10X
0,3 µL BSA 100X
1 µL XbaI
1 µL SpeI
22,7 µL water

Incubation 2h55 at 37°C and then 20 min at 65°C.

Electrophoresis

1% agarose gel
for PCR products: 10 µL loaded
for digestion products (30 µL): adding of 7 µL of loading blue and then 20 µL loaded on gel

well n°	1	2	3	4	5	6	7	8	9
method		PCR				digestion
sample	1 kb DNA ladder	positive control MP143	negative control	MP160.1	MP160.2	MP143	MP160.1	MP160.2	100 bp DNA ladder
expected size		1114 bp		1403 bp		876 bp (E0240) 2079 (pSB1A2)	1165 bp (FlhDC+promotor) 2079 bp (pSB1A2)
measured size		1 kb 3 kb		0,3 kb 1,8 kb	0,3 kb 1,8 kb	0,9 kb 2 kb	2 kb	2 kb

Results: The clones tested didn't have the insert.

Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240)

Digestion

Digestion

Protocol Digestion

Name	Template DNA	Description	Vol MP (µl)	Vol H2O (µl)	Enzymes
D168	MP143.1	RBS - GFP - term (FV)	7.25	17.45	EcoRI and XbaI

Gel Extraction

Gel Extraction of D168

Well	1	2	3	4	5	6
Sample	100 pb ladder	MP143.1	no sample	D168	no sample
Expected size (pb)				2 940
Measured size (pb)		5.000		3 000

Measurement of the concentration of D168 purified

Protocol (it's same that for Miniprep)

Digestion Name	Concentration (µg/mL)	Ratio 260/280
D168	24	2.15

Ligation

Ligation Name	Vector Name	Volume Vector (µL)	Insert	Volume Insert (µL)
L164	D168	1.67	D132	2.04
Control L164	D168	1.67	-	-

Construction for FIFO

Aim : Construction of pFlgA - YFP tripart (+/- LVA) "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)

Transformation of the ligations we did yesterday

Ligation Name	Description	Antibio
L160	D166 (FV) + D132 (FI) pFlgA - YFP tripart (LVA-)	Amp
TL160	D166 YFP tripart (LVA-)	Amp
L161	D167 (FV) + D132 (FI) pFlgA - YFP tripart (LVA+)	Amp
TL161	D167 YFP tripart (LVA+)	Amp

Construction for synchronization

Transformation of the ligations we did yesterday

Ligation Name	Description	Antibio
L158	D110 (BV) + D131 (BI)	Amp
Control L158	D110	Amp
L159	D125 (FV) + D109 (FI)	Kan
Control L159	D125	Kan

Ligation

Ligation Name	Description	Vector Name	Volume vector (µL)	Insert Name	Volume insert (µL)
L162	rbs-lasI + gfp generator	D107 (BV)	10	D163 (BI)	3.46
Control L162	autoligation control	D107 (BV)	10	-	-
L163	rbs-TetR + gfp generator	D110 (BV)	2.5	D163 (BI)	3.44
Control L163	autoligation control	D110 (BV)	2.5	-	-

Transformation

Ligation Name	Description	Antibio
L162	D107 (BV) + D163 (BI)	Amp
Control L162	D107	Amp
L163	D110 (BV) + D163 (BI)	Amp
Control L163	D110	Amp

Promoter characterization plasmids

Transformation of ligations from August 20th

Top 10 cells were used

Ligation name	Vector digestion	Vector description	C° Vector (µg/mL)	V. Vector (µl)	Insert digestion	Insert description	C° Insert (µg/mL)	V. Insert (µl)	Product description	Antibiotic
L155	D164	J23101 promoter	3	16	D163	gfp generator	14	8	J23101 promoter-gfp generator	Amp
L156	D161	pTet promoter	39	1.25	D163	gfp generator	14	5	pTet promoter-gfp generator	Kana
Control L156	D161			1.25	none				Vector autoligation control	Kana
L157	D125.2	B0015	15	3	D162	tetR	11	4	tetR-B0015	Kana
Control L157	D125.2			3	none				Vector autoligation control	Kana
L166	D185	RBS B0032	21	2	D182	tetR	10	6.5	RBS B0032 - tetR	Amp
Control L166	D185			2	none				Vector autoligation control	Amp
L167	D181	pTet	1	14	D184	gfp generator	14	3	gfp generator - pTet	Amp
Control L167	D181	pTet		14	none				Vector autoligation control	Amp

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