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	|2		|2
	|pJ23107>>lox71-gfptripart(T)		|pJ23107>>lox71-gfptripart(T)
		+	|
		+	|- style="background: #cccccc;"
		+	| style="background: #ccffcc;" |L52
		+	|RBSDapAcoli BI (D63)
		+	|10
		+	|gfptripart-lox66 BV <bbpart>pSB1A2</bbpart> (D69)
		+	|2
		+	|gfptripart-lox66-RBSDapAcoli
		+	|
		+	|- style="background: #cccccc;"
		+	| style="background: #ccffcc;" |L53
		+	|RBSDapAcoli BI (D63)
		+	|10
		+	|B0015(T)-lox66 BV (D58.1)
		+	|3
		+	|gfptripart-lox66-RBSDapAcoli
		+	|
		+	|- style="background: #cccccc;"
		+	| style="background: #ccffcc;" |L54
		+	|RBSDapAcoli BI (D63)
		+	|10
		+	|attB BV (D68)
		+	|2
		+	|attB-lox66-RBSDapAcoli
		+	|
		+	|- style="background: #cccccc;"
		+	| style="background: #ccffcc;" |L55
		+	|RBSdapAsubtilis BI (D54)
		+	|15
		+	|gfptripart-lox66 BV <bbpart>pSB1A2</bbpart> (D69)
		+	|2
		+	|gfptripart-lox66-RBSdapAsubtilis
		+	|
		+	|- style="background: #cccccc;"
		+	| style="background: #ccffcc;" |L56
		+	|RBSdapAsubtilis BI (D54)
		+	|15
		+	|B0015(T)-lox66 BV (D58.1)
		+	|3
		+	|gfptripart-lox66-RBSdapAsubtilis
		+	|
		+	|- style="background: #cccccc;"
		+	| style="background: #ccffcc;" |L57
		+	|RBSdapAsubtilis BI (D54)
		+	|15
		+	|attB BV (D68)
		+	|2
		+	|attB-lox66-RBSdapAsubtilis
	|		|
	|}		|}

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Revision as of 19:06, 20 September 2007

Contents 1 DapA Wanner deletion: pKD13-DapA homologies PCR 2 Minipreps 3 Digestion reactions 4 Ligation reactions

DapA Wanner deletion: pKD13-DapA homologies PCR

We diluted 1/100 overnight culture at 30°C of MG1655 transformed by pKD46: 50µL into 5mL LB ampicilline.
At OD = 0.1, we induced pKD46 expression by addition of arabinose (10mM final).
At OD = 0.4, we prepared cells to be electrocompetent. (see Protocol)
Then we transformed the bacteria with PCR product from yesterday (see September 17).

Minipreps

Minipreps sur cultures ON de:

• S24: (lox66-DapAsubtilis) MPT3.? (préciser David)
• S26: (lRBS-DapAsubtilis) MPT5.? (préciser David)
• pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé

Digestion reactions

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description
D77	araC/pBad>>lox71-gfptripart BI	D60 (.1 & .2) digestion product	FspI	AhdI		in D60, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion
D78	araC/pBad>>lox71-gfptripart FI	D71 (.1 & .2) digestion product	FspI	AhdI		in D71, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion
D53	RBS-dapAsubtilis BBinsert	pTOPO of RBS-dapAsubtilis (MPT5.2)	EcoRI	Pst1		RBS-dapAsubtilis extracted for cloning as a biobrick
D54	RBS-dapAsubtilis BI	pTOPO of RBS-dapAsubtilis (MPT5.2)	XbaI	Pst1		RBS-dapAsubtilis extracted for use as a BI

Ligation reactions

Ligations
Number	Insert	Insert Volume (µL)	Vector	Vector Volume (µL)	Comments	Number of colonies
C10			gfptripart-lox66 BV pSB1A2 (D69)	2
C11			B0015(T)-lox66 BV (D58.1)	3
C12			attB BV (D68)	2
C13			FRT-CmR-FRT BBa_J61025 BV (D64)	1.5
C14			lox66-mRFP FV (D61.1)	1.5
C15			B0015 BV pSB1AK3 (D46)	1
C16			pSB2K3 ES open vector (D73)	4
C17			pSB2K3 BBvector (D72)	2
C18			pSB1A2 EP open vector (D22)	2
L44	lox71-B0015(T) BI (D57.1)	15	pJ23100 SP(D1)	2	pJ23100>>lox71-B0015(T)
L45	lox71-B0015(T) BI (D57.1)	15	pJ23107 SP(D3)	2	pJ23107>>lox71-B0015(T)
L46	lox71-B0015(T) BI (D57.2)	15	BBa_J61002 pTet SP dig.(D50)	2	pTet>>lox71-B0015(T)
L47	lox71-B0015(T) BI (D57.2)	15	araC/pBad-J61002 plasmid SP dig.(D41)	2	araC/pBad>>lox71-B0015(T)
L48	lox71-gfp(tripart) BI (D37.1)	10	pJ23100 SP(D1)	2	pJ23100>>lox71-gfptripart
L49	lox71-gfp(tripart) BI (D37.1)	10	pJ23107 SP(D3)	2	pJ23107>>lox71-gfptripart(T)
L52	RBSDapAcoli BI (D63)	10	gfptripart-lox66 BV pSB1A2 (D69)	2	gfptripart-lox66-RBSDapAcoli
L53	RBSDapAcoli BI (D63)	10	B0015(T)-lox66 BV (D58.1)	3	gfptripart-lox66-RBSDapAcoli
L54	RBSDapAcoli BI (D63)	10	attB BV (D68)	2	attB-lox66-RBSDapAcoli
L55	RBSdapAsubtilis BI (D54)	15	gfptripart-lox66 BV pSB1A2 (D69)	2	gfptripart-lox66-RBSdapAsubtilis
L56	RBSdapAsubtilis BI (D54)	15	B0015(T)-lox66 BV (D58.1)	3	gfptripart-lox66-RBSdapAsubtilis
L57	RBSdapAsubtilis BI (D54)	15	attB BV (D68)	2	attB-lox66-RBSdapAsubtilis