Paris/September 18

From 2007.igem.org

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 +++