Paris/September 18
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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.
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 | +++ |
