From 2013.igem.org

Experiment > 4th week

September 22th
Mini prep following plasmid.

fimE-pBAD(in pSB1A3), pSB1C3-Km, pSB1A3-Km

Result.

1: pSB1C3-Km, 2-4: pSB1A3-Km, 5-6: fimE-pBAD

Check PCR.

Fragment	(control)	Fragment 3 - ApCm
Primer	141-49 SaApL	141-49 SaApL
Templete	Escherichia coli MG1655 red (Cell suspention)	(Cell suspention)

Result.

1-9: Fragment 3 - ApCm

Check PCR.

Fragment	(control)	Fragment 3 - ApCm	Fragment 3 - ApCm
Primer	2 sets of the others	141-1 141-20	141-1 141-74
Templete	Escherichia coli MG1655 red (Cell suspention)	(Cell suspention)

Result.

1-7: 141-1 and 141-20, 8: control(141-1 and 141-20),
9-15: 141-1 and 141-74, 16: control(141-1 and 141-74),

Digestion following plasmid.

pSB1C3-Km-Blunt

P1 Preparation following fragment.

Fragment 1, 2

September 23th
PCR following fragment.

Fragment	Fragment 3 - ApCm
Primer	mhpA-CmC6 SaApL
Templete	Fragment 3 - Cm Ap(SaApR-SaApL)

Result.

1: Fragment 3 - Cm, 2: Fragment 3 - ApCm,
3: pSB1C3-Km-Blunt

Check PCR.

Fragment	Fragment 3 - Ap (control)	Fragment 3 - ApCm
Primer	CmNR-Not SaApL	CmNR-Not SaApL
Templete	(Cell suspention)	(Cell suspention)

Result.

1-31: Fragment 3 - ApCm

Electro Pration

Fragment 3 - Cm to Escherichia coli MG1655 red.

P1 Preparation

Fragment 3 - ApCm

September 24th
P1 transdunction

Fragment 1, 2

Result.

Fragment	Fragment 3 - Ap (control)	Fragment 3 - ApCm
Primer	CmNR-Not 141-1	CmNR-Not 141-1
Templete	(Cell suspention)	(Cell suspention)

Result.

1-16: Fragment 3 - ApCm

P1 transduction

Fragment 2 to Fragment 1

Fragment 1 to Fragment 2

Result.

Get Fragment 1-2

Electro Poration

Fragment 1, 2, 3 - Ap, 3 - Cm, 3 - ApCm (in pSBC3-Km) to Escherichia coli DH1

Result.

September 25th
Check PCR.

Fragment	Fragment 3 - ApCm (control)	Fragment 3 - ApCm
Primer	141-49 141-1	CmNR-Not 141-1
Templete	(Cell suspention)	(Cell suspention)

Result.

1-7: 141-49 and 141-1, 8: control(141-49 and 141-8),
9-15: 141-1 and CmNR-Not, 16: control(141-1 and CmNR-Not)

September 26th
P1 transduction

Fragment 3 to Fragment 1-2

Get Fragment 1-2-3.

Check PCR

Fragment	Fragment 1-2-3	Fragment 1-2 (control)
Primer	CmNR-Not 141-1	141-49 141-1
Templete	(Cell suspention)	(Cell suspention)

Result.

1-7: Fragment 1-2-3, 8: cotrol, 9-10: Fragment 1,
11-13: Fragment 2, 14-15: Fragment 3 - Ap, 16-17: Fragment 3 - Cm
18-19: Fragment 3 - AmCm

September 27th
Assay
Pythagorean devices

LB 2ml + arabinose -10% 0.1ml	+ with the inoculating loop 1655 ins①~③ No.1
LB 2ml + arabinose -10% 0.1ml	+ with the inoculating loop 1655 ins①~③ No.2
LB 2ml + arabinose -10% 0.1ml	+ with the inoculating loop 1655 ins①~③ No.3
LB 2ml + arabinose -10% 0.1ml	+ 1655
LB 2ml	+ with the inoculating loop 1655 ins①~③ No.1
LB 2ml	+ with the inoculating loop 1655 ins①~③ No.2
LB 2ml	+ with the inoculating loop 1655 ins①~③ No.3
LB 2ml	+ 1655

⇒I culture it at 39 degrees for 2h.

ara(+) No.1~3 ara(-) No.1~3 ara(+) ara(-)

Premix(1)	Cell suspension	ins①-③
		Ins①-③
		1655
		1655
	GXL Buff	20
	dNTP	8
	GXL	2
		(30)
Premix(2)	Premix(1)	27
	CmNR-not2	9
	141-1	9
		(45)
mix	Cell suspension	5λ
	Premix(2)	5λ
		<4P3-3>

No.1~3 No.1~3<

Cell suspension	1655	ins①~③	ara(+)
	1655	ins①~③	ara(-)
Premix①	GXL Buffer	30
	dNTP	12
	GXL	3
		(45)
Premix②
	Premix(1)	21	21
	FimE-C2-EcoRI	7	7
	TetP1	7
	TetP2		7
		(35)	(35)
mix	Premix②	5
	Cell suspension	5
	<4P3-3>

LB + arabinose (0.2%)	→	plating (single colony isolation)
	→	1655 + ins①~③ No.1~3
	→	1655 No.1~3
LB + IPTG (0.4g/ml)	→	plating (single colony isolation)
	→	1655 + ins①~③ No.1~3
	→	1655 No.1~3
LB	→	plating (single colony isolation)
	→	1655 + ins①~③ No.1~3
	→	1655 No.1~3

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Team:TMU-Tokyo/Notebook experiment/4th

From 2013.igem.org

Experiment > 4th week