Paris/September 18

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[[Paris/September 17|yesterday]] -- [[Paris/September 19|tomorrow]] <br>
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== 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.<br>
 +
At OD = 0.1, we induced pKD46 expression by addition of arabinose (10mM final). <br>
 +
At OD = 0.4, we prepared cells to be electrocompetent. (see [[Paris/PROTOCOLS#Recombineering.2FLambda_red-mediated_gene_replacement|Protocol]])<br>
 +
Then we transformed the bacteria with PCR product from yesterday (see [[Paris/September_17|September 17]]).
 +
== Minipreps ==
== Minipreps ==
Line 6: Line 14:
*S26: (lRBS-DapAsubtilis) MPT5.? (préciser David)
*S26: (lRBS-DapAsubtilis) MPT5.? (préciser David)
*pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé
*pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé
-
 
== Digestion reactions ==
== Digestion reactions ==
Line 28: Line 35:
|araC/pBad>>lox71-gfptripart BI
|araC/pBad>>lox71-gfptripart BI
|D60 (.1 & .2) digestion product
|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
|in D60, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion
Line 36: Line 43:
|
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #FF99FF;" |D78
+
| style="background: #ccffcc;" |D78
|araC/pBad>>lox71-gfptripart FI
|araC/pBad>>lox71-gfptripart FI
|D71 (.1 & .2) digestion product
|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
|in D71, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion
Line 68: Line 75:
|
|
|
|
 +
|}
 +
 +
 +
 +
== Ligation reactions ==
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Ligations
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=20%| Insert
 +
|width=5%| Insert Volume (µL)
 +
|width=25%| Vector
 +
|width=5%| Vector Volume (µL)
 +
|width=35%| Comments
 +
|width=5%| Number of colonies
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C10
 +
|
 +
|
 +
|gfptripart-lox66 BV <bbpart>pSB1A2</bbpart> (D69)
 +
|2
 +
|
 +
| +++
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C11
 +
|
 +
|
 +
|B0015(T)-lox66 BV (D58.1)
 +
|3
 +
|
 +
| +++
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C12
 +
|
 +
|
 +
|attB BV (D68)
 +
|2
 +
|
 +
|110
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C13
 +
|
 +
|
 +
|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> BV (D64)
 +
|1.5
 +
|
 +
| +++
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C14
 +
|
 +
|
 +
|lox66-mRFP FV (D61.1)
 +
|1.5
 +
|
 +
|150
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C15
 +
|
 +
|
 +
|<bbpart>B0015</bbpart> BV <bbpart>pSB1AK3</bbpart> (D46)
 +
|1
 +
|
 +
|1
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C16
 +
|
 +
|
 +
|<bbpart>pSB2K3</bbpart> ES open vector (D73)
 +
|4
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C17
 +
|
 +
|
 +
|<bbpart>pSB2K3</bbpart> BBvector (D72)
 +
|2
 +
|
 +
|1
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C18
 +
|
 +
|
 +
|pSB1A2 EP open vector (D22)
 +
|2
 +
|
 +
|16
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L44
 +
|lox71-B0015(T) BI (D57.1)
 +
|15
 +
|<bbpart>pJ23100</bbpart> SP(D1)
 +
|2
 +
|pJ23100>>lox71-B0015(T)
 +
|18
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L45
 +
|lox71-B0015(T) BI (D57.1)
 +
|15
 +
|<bbpart>pJ23107</bbpart> SP(D3)
 +
|2
 +
|pJ23107>>lox71-B0015(T)
 +
|30
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L46
 +
|lox71-B0015(T) BI (D57.2)
 +
|15
 +
|<bbpart>BBa_J61002</bbpart> pTet SP dig.(D50)
 +
|2
 +
|pTet>>lox71-B0015(T)
 +
| +++
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L47
 +
|lox71-B0015(T) BI (D57.2)
 +
|15
 +
|araC/pBad-J61002 plasmid SP dig.(D41)
 +
|2
 +
|araC/pBad>>lox71-B0015(T)
 +
| +++
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L48
 +
|lox71-gfp(tripart) BI (D37.1)
 +
|10
 +
|<bbpart>pJ23100</bbpart> SP(D1)
 +
|2
 +
|pJ23100>>lox71-gfptripart
 +
|1
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L49
 +
|lox71-gfp(tripart) BI (D37.1)
 +
|10
 +
|<bbpart>pJ23107</bbpart> SP(D3)
 +
|2
 +
|pJ23107>>lox71-gfptripart(T)
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L52
 +
|RBSDapAcoli BI (D63)
 +
|10
 +
|gfptripart-lox66 BV <bbpart>pSB1A2</bbpart> (D69)
 +
|2
 +
|gfptripart-lox66-RBSDapAcoli
 +
|~>C10
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L53
 +
|RBSDapAcoli BI (D63)
 +
|10
 +
|B0015(T)-lox66 BV (D58.1)
 +
|3
 +
|B0015(T)-lox66-RBSDapAcoli
 +
|>C11
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L54
 +
|RBSDapAcoli BI (D63)
 +
|10
 +
|attB BV (D68)
 +
|2
 +
|attB-lox66-RBSDapAcoli
 +
|>C12
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L55
 +
|RBSdapAsubtilis BI (D54)
 +
|15
 +
|gfptripart-lox66 BV <bbpart>pSB1A2</bbpart> (D69)
 +
|2
 +
|gfptripart-lox66-RBSdapAsubtilis
 +
|~>C10
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L56
 +
|RBSdapAsubtilis BI (D54)
 +
|15
 +
|B0015(T)-lox66 BV (D58.1)
 +
|3
 +
|B0015(T)-lox66-RBSdapAsubtilis
 +
|>C11
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L57
 +
|RBSdapAsubtilis BI (D54)
 +
|15
 +
|attB BV (D68)
 +
|2
 +
|attB-lox66-RBSdapAsubtilis
 +
|>C12
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L58
 +
|pTet>>lox71-gfptripart BI (D59)
 +
|15.5
 +
|FRT-CmR-FRT <bbpart>BBa_J61025</bbpart> BV (D64)
 +
|1.5
 +
|FRT-CmR-FRT-pTet>>lox71-gfptripart
 +
|=C13
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L59
 +
|pTet>>lox71-gfptripart FI (D70)
 +
|15.5
 +
|lox66-mRFP FV (D61.1)
 +
|1.5
 +
|pTet>>lox71-gfptripart-lox66-mRFP
 +
|>C14
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L60
 +
|lox66-RBSDapAcoli BI (D62)
 +
|16
 +
|<bbpart>B0015</bbpart> BV <bbpart>pSB1AK3</bbpart> (D46)
 +
|1
 +
|B0015(T)-lox66-RBSDapAcoli
 +
|~50
 +
|- style="background: #cccccc;" 
 +
| 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;" |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;" |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>
 +
| +++
|}
|}

Latest revision as of 18:05, 7 October 2007

yesterday -- tomorrow

Contents

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 +++
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