Paris/September 18

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Latest revision as of 18:05, 7 October 2007

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		110
C13			FRT-CmR-FRT BBa_J61025 BV (D64)	1.5		+++
C14			lox66-mRFP FV (D61.1)	1.5		150
C15			B0015 BV pSB1AK3 (D46)	1		1
C16			pSB2K3 ES open vector (D73)	4		0
C17			pSB2K3 BBvector (D72)	2		1
C18			pSB1A2 EP open vector (D22)	2		16
L44	lox71-B0015(T) BI (D57.1)	15	pJ23100 SP(D1)	2	pJ23100>>lox71-B0015(T)	18
L45	lox71-B0015(T) BI (D57.1)	15	pJ23107 SP(D3)	2	pJ23107>>lox71-B0015(T)	30
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	1
L49	lox71-gfp(tripart) BI (D37.1)	10	pJ23107 SP(D3)	2	pJ23107>>lox71-gfptripart(T)	0
L52	RBSDapAcoli BI (D63)	10	gfptripart-lox66 BV pSB1A2 (D69)	2	gfptripart-lox66-RBSDapAcoli	~>C10
L53	RBSDapAcoli BI (D63)	10	B0015(T)-lox66 BV (D58.1)	3	B0015(T)-lox66-RBSDapAcoli	>C11
L54	RBSDapAcoli BI (D63)	10	attB BV (D68)	2	attB-lox66-RBSDapAcoli	>C12
L55	RBSdapAsubtilis BI (D54)	15	gfptripart-lox66 BV pSB1A2 (D69)	2	gfptripart-lox66-RBSdapAsubtilis	~>C10
L56	RBSdapAsubtilis BI (D54)	15	B0015(T)-lox66 BV (D58.1)	3	B0015(T)-lox66-RBSdapAsubtilis	>C11
L57	RBSdapAsubtilis BI (D54)	15	attB BV (D68)	2	attB-lox66-RBSdapAsubtilis	>C12
L58	pTet>>lox71-gfptripart BI (D59)	15.5	FRT-CmR-FRT BBa_J61025 BV (D64)	1.5	FRT-CmR-FRT-pTet>>lox71-gfptripart	=C13
L59	pTet>>lox71-gfptripart FI (D70)	15.5	lox66-mRFP FV (D61.1)	1.5	pTet>>lox71-gfptripart-lox66-mRFP	>C14
L60	lox66-RBSDapAcoli BI (D62)	16	B0015 BV pSB1AK3 (D46)	1	B0015(T)-lox66-RBSDapAcoli	~50
L61	AraC/pBAD>>RBS-CRE FI (D75)	13	pSB2K3 ES open vector (D73)	4	AraC/pBAD>>RBS-CRE cloned in pSB2K3	0
L62	araC/pBad>>RBS-Cre BBinsert (D56)	15	pSB2K3 BBvector (D72)	2	AraC/pBAD>>RBS-CRE cloned in pSB2K3	0
L63	DGAT BB (D76)	15	pSB1A2 EP open vector (D22)	2	DGAT cloned in pSB1A2	12
L64	RBSdapAsubtilis BBinsert (DD53)	15	pSB1A2 EP open vector (D22)	2	RBSdapAsubtilis cloned in pSB1A2	+++

@@ Line 1: / Line 1: @@
+[[Paris/September 17|yesterday]] -- [[Paris/September 19|tomorrow]] <br>
 == DapA Wanner deletion: pKD13-DapA homologies PCR ==
@@ Line 97: / Line 98: @@
 |2
 |
-|
+| +++
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C11
@@ Line 105: / Line 106: @@
 |3
 |
-|
+| +++
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C12
@@ Line 113: / Line 114: @@
 |2
 |
-|
+|110
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C13
@@ Line 121: / Line 122: @@
 |1.5
 |
-|
+| +++
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C14
@@ Line 129: / Line 130: @@
 |1.5
 |
-|
+|150
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C15
@@ Line 137: / Line 138: @@
 |1
 |
-|
+|1
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C16
@@ Line 145: / Line 146: @@
 |4
 |
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C17
@@ Line 153: / Line 154: @@
 |2
 |
-|
+|1
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |C18
@@ Line 161: / Line 162: @@
 |2
 |
-|
+|16
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L44
@@ Line 169: / Line 170: @@
 |2
 |pJ23100>>lox71-B0015(T)
-|
+|18
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L45
@@ Line 177: / Line 178: @@
 |2
 |pJ23107>>lox71-B0015(T)
-|
+|30
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L46
@@ Line 185: / Line 186: @@
 |2
 |pTet>>lox71-B0015(T)
-|
+| +++
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L47
@@ Line 193: / Line 194: @@
 |2
 |araC/pBad>>lox71-B0015(T)
-|
+| +++
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L48
@@ Line 201: / Line 202: @@
 |2
 |pJ23100>>lox71-gfptripart
-|
+|1
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L49
@@ Line 209: / Line 210: @@
 |2
 |pJ23107>>lox71-gfptripart(T)
-|
+|0
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L52
@@ Line 217: / Line 218: @@
 |2
 |gfptripart-lox66-RBSDapAcoli
-|
+|~>C10
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L53
@@ Line 224: / Line 225: @@
 |B0015(T)-lox66 BV (D58.1)
 |3
-|gfptripart-lox66-RBSDapAcoli
+|B0015(T)-lox66-RBSDapAcoli
-|
+|>C11
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L54
@@ Line 233: / Line 234: @@
 |2
 |attB-lox66-RBSDapAcoli
-|
+|>C12
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L55
@@ Line 241: / Line 242: @@
 |2
 |gfptripart-lox66-RBSdapAsubtilis
-|
+|~>C10
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L56
@@ Line 248: / Line 249: @@
 |B0015(T)-lox66 BV (D58.1)
 |3
-|gfptripart-lox66-RBSdapAsubtilis
+|B0015(T)-lox66-RBSdapAsubtilis
-|
+|>C11
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L57
@@ Line 257: / Line 258: @@
 |2
 |attB-lox66-RBSdapAsubtilis
-|
+|>C12
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L58
@@ Line 265: / Line 266: @@
 |1.5
 |FRT-CmR-FRT-pTet>>lox71-gfptripart
-|
+|=C13
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L59
@@ Line 273: / Line 274: @@
 |1.5
 |pTet>>lox71-gfptripart-lox66-mRFP
-|
+|>C14
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |L60
@@ Line 281: / Line 282: @@
 |1
 |B0015(T)-lox66-RBSDapAcoli
-|
+|~50
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |C16
+| style="background: #ccffcc;" |L61
-|
+|AraC/pBAD>>RBS-CRE FI (D75)
-|
+|13
 |<bbpart>pSB2K3</bbpart> ES open vector (D73)
 |4
-|
+|AraC/pBAD>>RBS-CRE cloned in <bbpart>pSB2K3</bbpart>
-|
+|0
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |C17
+| style="background: #ccffcc;" |L62
-|
+|araC/pBad>>RBS-Cre BBinsert (D56)
-|
+|15
 |<bbpart>pSB2K3</bbpart> BBvector (D72)
 |2
-|
+|AraC/pBAD>>RBS-CRE cloned in <bbpart>pSB2K3</bbpart>
-|
+|0
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |C18
+| style="background: #ccffcc;" |L63
-|
+|DGAT BB (D76)
-|
+|15
 |pSB1A2 EP open vector (D22)
 |2
-|
+|DGAT cloned in <bbpart>pSB1A2</bbpart>
-|
+|12
+|- style="background: #cccccc;"
+| style="background: #ccffcc;" |L64
+|RBSdapAsubtilis BBinsert (DD53)
+|15
+|pSB1A2 EP open vector (D22)
+|2
+|RBSdapAsubtilis cloned in <bbpart>pSB1A2</bbpart>
+| +++
 |}

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